Low molecular weight heparin composition and uses thereof

ABSTRACT

Preparations of low molecular weight heparins (LMWHs) having improved properties, e.g., properties that provide a clinical advantage, are provided herein. Methods of making and using such preparations as well as methods of analyzing starting materials, processing, intermediates and final products in the production of such LMWH preparations are provided.

This application is a divisional of U.S. application Ser. No. 11/805,829filed May 24, 2007 which claims priority to U.S. Provisional ApplicationSer. Nos. 60/809,136, filed, on May 25, 2006; 60/849,578, filed on Oct.4, 2006; and 60/849,628, filed on Oct. 5, 2006, the contents of whichare incorporated herein by reference.

BACKGROUND

Coagulation is a physiological pathway involved in maintaining normalblood hemostasis in mammals. Under conditions in which a vascular injuryoccurs, the coagulation pathway is stimulated to form a blood clot toprevent the loss of blood. Immediately after the vascular injury occurs,blood platelets begin to aggregate at the site of injury forming aphysical plug to stop the leakage. In addition, the injured vesselundergoes vasoconstriction to reduce the blood flow to the area andfibrin begins to aggregate forming an insoluble network or clot, whichcovers the ruptured area.

When an imbalance in the coagulation pathway shifts towards excessivecoagulation, the result is the development of thrombotic tendencies,which are often manifested as heart attacks, strokes, deep veinthrombosis, and acute coronary syndromes such as myocardial infarcts,and unstable angina. Furthermore, an embolism can break off from athrombus and result in a pulmonary embolism or cerebral vascularembolism including stroke or transient ischemia attack. Currenttherapies for treating disorders associated with imbalances in thecoagulation pathway involve many risks and must be carefully controlled.

Heparin and low molecular weight heparins (LMWHs), complex, sulfatedpolysaccharides isolated from endogenous sources, are potent modulatorsof hemostasis. Heparin, a highly sulfated heparin-like glycosaminoglycan(HLGAG) produced by mast cells, is a widely used clinical anticoagulant,and is one of the first biopolymeric drugs and one of the fewcarbohydrate drugs. Heparin and molecules derived from it are potentanticoagulants that are used in a variety of clinical situations,especially for thromboembolic disorders including the prophylaxis andtreatment of deep venous thrombosis and pulmonary embolism, arterialthromboses, and acute coronary syndromes like myocardial infarction andunstable angina. Heparin and LMWHs interact with multiple components ofthe coagulation cascade to inhibit the clotting process. Heparinprimarily elicits its effect through two mechanisms, both of whichinvolve binding of antithrombin III (AT-III) to a specificpentasaccharide sequence, H_(NAc/S,6S)GH_(NS,3S,6S)I_(2S)H_(NS,6S)contained within the polymer. First, AT-III binding to thepentasaccharide induces a conformational change in the protein thatmediates its inhibition of factor Xa. Second, thrombin (factor IIa) alsobinds to heparin at a site proximate to the pentasaccharide/AT-IIIbinding site. Formation of a ternary complex between AT-III, thrombinand heparin results in inactivation of thrombin. Unlike its anti-Xaactivity that requires only the AT-III pentasaccharide-binding site,heparin's anti-IIa activity is size-dependent, in addition to thepentasaccharide unit responsible for anti-Xa activity for the efficientformation of an AT-III, thrombin, and heparin ternary complex. Heparinalso mediates the release of tissue factor pathway inhibitor (TFPI) fromendothelial cells. TFPI, a heparin cofactor, is a serine protease thatdirectly binds to and inhibits factor X. TFPI is a potentanti-thrombotic, particularly when co-administered with heparin.

Although heparin is highly efficacious in a variety of clinicalsituations and has the potential to be used in many others, the sideeffects associated with heparin therapy are many and varied.Anti-coagulation has been the primary clinical application forunfractionated heparin (UFH) for over 65 years. Due to its erraticintravenous pharmacokinetics and lack of subcutaneous bioavailability,UFH has been administered by intravenous injection instead.Additionally, the application of UFH as an anticoagulant has beenhampered by the many side effects associated with non-specific plasmaprotein binding with UFH.

This has led to the explosion in the generation and utilization of lowmolecular weight heparin (LMWH) as an efficacious alternative to UFH.LMWH provide a more predictable pharmacological action, reduced sideeffects, and better bioavailability than UFH. Since the commerciallyavailable LMWH preparations are not fully neutralized by protamine, anunexpected reaction could have extremely adverse effects; the anti-Xaactivity of enoxaparin and other LMWH are neutralizable only to anextent of about 40% with ≦2 mg Protamine/100 IU anti-Xa LMWH. Theanti-IIa activity is neutralizable only to an extent of about 60% with≦2 mg Protamine/100 IU anti-Xa LMWH. (On the other hand, the anti-Xa andanti-IIa activity of UFH is neutralizable almost completely (>90%) with≦2 mg Protamine sulfate/100 IU anti-Xa UFH.)

Pharmaceutical preparations of these polysaccharides, typically isolatedfrom porcine intestinal mucosa, are heterogeneous in length andcomposition. As such, only a portion of a typical preparation possessesanticoagulant activity. At best, the majority of the polysaccharidechains in a pharmaceutical preparation of heparin or LMWH are inactive,at worst, these chains interact nonspecifically with plasma proteins toelicit the side effects associated with heparin therapy. Therefore, itis important to develop novel LMWHs that retain the anticoagulantactivity and other desired activities of UFH but have reduced sideeffects. LMWHs, essentially due to their reduced chains sizes anddispersity, display markedly less non-specific plasma protein binding.However, all LMWHs that are currently clinically available also possessreduced anti-IIa activity as compared to UFH. Because of this decreasedactivity, a larger dose of LMWH is required (compared to UFH) in orderto achieve a similar anti-Xa and anti-IIa activity, and the standardtests for UFH activity, activated partial thromboplastin time (aPTT) oractivated clotting time (ACT), are not useful as they rely primarily onanti-IIa activity for a readout. The most widely used test formonitoring LMWH levels is an anti-Xa activity test, which depends on thesubject having sufficient levels of antithrombin III (ATIII), which isnot always the case. This test is quite costly (well over $100.00) andis not routine or readily available, as samples generally must be sentto an outside lab for analysis. Consequently, the use of LMWHs so farhas been largely limited to the prevention of thrombosis and not totheir treatment, and the population of patients to whom it can beadministered has been limited, excluding, among others, pediatricpatients, patients with abnormal renal function as measured by RFI,urea, creatinine, phosphorus, glomerular filtration rate (GFR), or BUN(Blood Urea Nitrogen level) in blood and urine and the interventionalcardiology patient population.

SUMMARY OF THE INVENTION

The invention is based, in part, on the development of preparations ofLMWHs having, e.g., designed to have, improved properties, e.g.,properties that provide a clinical advantage. Such functional propertiesinclude, by way of example, one or more of: reversibility in response toprotamine sulfate; predictable or otherwise improved pharmacokinetics;improved anti-IIa activity, as compared, e.g., to enoxaparin; arelatively constant anti-Xa activity to anti-IIa activity ratio over aperiod of about 30 to 180 minutes; monitorable activity levels;subcutaneous bioavailability; and reduced likelihood of causing heparininduced thrombocytopenia (HIT). LMWHs disclosed herein can also havestructural characteristics that distinguish them from other commerciallyavailable LMWHs. For example, a LMWH preparation provided herein canhave one or more of the following characteristics: substantiallyundetectable linkage region; an increased amount of 3-O sulfates ascompared to commercially available LMWH preparations; a subset of thechains have an unsulfated ΔU at the non-reducing end; a subset of thechains, e.g., a majority, e.g., substantially all of the chains, have anN-acetylated hexosamine at the reducing end; a ratio ofΔUH_(NAc,6S)GH_(Ns,3S,6S) to ΔU_(2S)H_(NS,6S)IH_(NAc,6S)GH_(NS,3S,6S) ofabout 1:1 to 4:1 (e.g., about 1:1, 2:1, 3:1, 4:1), and substantially nomodified reducing end structures. The invention includes LMWHpreparations having one or more of these properties and characteristicsas well as methods of making and using such preparations. The inventionalso features methods of analyzing starting materials, processing,intermediates and final products in the production of such LMWHpreparations.

Accordingly, in a first aspect, the invention features, a LMWHcomposition having: a weight average molecular weight of about 5000 to9000 Da, e.g., about 5000 to 8300 Da, e.g., about 5500 to 8000 Da, e.g.,about 5700 to 7900 Da, e.g., about 5800 to 6800 Da; and

an anti-IIa activity of about 50 to 300, e.g., about 70 to 280, e.g.,about 90 to 250 IU/mg, e.g., about 100 to 250 IU/mg, e.g., about 100 to140 IU/mg, 150 to 200 IU/mg, about 130 to 190 IU/mg, e.g., about 155 to195 IU/mg.

In a second aspect, the invention features, a LMWH composition having:

a weight average molecular weight of about 5000 to 9000 Da, e.g., about5000 to 8300 Da, e.g., about 5000 to 8000 Da, about 5500 to 8000 Da,e.g., about 5700 to 7900 Da, e.g., about 5800 to 6800 Da; and

anti-IIa activity that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%,99% or 100% neutralizable with protamine, e.g., as measured by activatedpartial thromboplastin time (ACT) or activated partial thromboplastintime (aPTT). Preferably, the anti-IIa activity of the LMWH isneutralized by at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100%within 5, 10, 15, 30 minutes after protamine administration.

In a third aspect, the invention features, a LMWH composition having: aweight average molecular weight of about 5000 to 9000 Da, e.g., about5000 to 8300 Da, e.g., about 5500 to 8000 Da, e.g., about 5700 to 7900Da, e.g., about 5800 to 6800 Da; and

ΔUH_(NAc,6S)GH_(NS,3S,6S) is 5 to 15%, e.g., 7 to 14%, e.g., 9 to 12%,of the composition, e.g., as measured by mole %. Preferably theΔUH_(NAc,6S)GH_(NS,3S,6S) at the non-reducing end of the molecule ofabout 5 to 15%, e.g., 7 to 14%, e.g., 9 to 12%, of the chains in thecomposition, e.g., as measured by mole %.

In a fourth aspect, the invention features, a LMWH composition having:

an average chain length of about 9 to 18 disaccharides or 8 to 18disaccharides, e.g., about 9 to 16 or 8 to 16 disaccharides; and

ΔUH_(NAc,6S)GH_(NS,3S,6S) is 5 to 15%, e.g., 7 to 14%, e.g., 9 to 12%,of the composition, e.g., as measured by mole %. Preferably theΔUH_(NAc,6S)GH_(NS,3S,6S) at the non-reducing end of the molecule ofabout 5 to 15%, e.g., 7 to 14%, e.g., 9 to 12%, of the chains in thecomposition, e.g., as measured by mole %.

In a fifth aspect, the invention features, a LMWH composition having:

a weight average molecular weight of 5000 to 9000 Da, e.g., about 5000to 8300 Da, e.g., about 5500 to 8000 Da, e.g., about 5700 to 7900 Da,e.g., about 5800 to 6800 Da; and

an anti Xa to anti-IIa ratio of 3:1 or less, e.g., 2:1, e.g., 1.6:1,1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1 or 0.5:1.

Preferably, the anti-Xa to anti-IIa ratio remains relatively constantover the course of an administration of the LMWH preparation, e.g., theanti-Xa to anti-IIa ratio varies no more than about +1.5, +1, ±0.5, or±0.2, over a period of about 30, 60, 120, 180, 240, 300 minutes. Forexample, if an initial ratio of anti-Xa activity to anti-IIa activity is2, then the ratio measured at a second time (e.g., 30, 60, 120, 180,240, 300 minutes) after the initial administration will preferably beless than 3, and preferably at or around 2.

In a seventh aspect, the invention features, a LMWH composition having:

optionally, a weight average molecular weight of about 5000 to 9000 Da,e.g., about 5000 to 8300 Da, e.g., about 5500 to 8000 Da, e.g., about5700 to 7900 Da, e.g., about 5800 to 6800 Da; and

when analyzed by digestion with heparinase I, heparinase II andheparinase III and capillary electrophoresis, each of peaks 1-14 ofTable 10A is present.

In a preferred embodiment: the amount of each peak in the LMWHcomposition, as analyzed by digestion with heparinase I, heparinase IIand heparinase III and capillary electrophoresis is about that found inTable 10A, the amount of each peak is within a range provided in Table10A; the amount of peaks 10 and 11 are within a range provided in Table10A.

In an eighth aspect, the invention features, a LMWH composition having:

optionally, a weight average molecular weight of about 5000 to 9000 Da,e.g., about 5000 to 8300 Da, e.g., about 5500 to 8000 Da, e.g., about5700 to 7900 Da, e.g., about 5800 to 6800 Da; and

when analyzed by 2D nuclear magnetic resonance (NMR) protons for each ofthe structures of Table 11A are present.

In a preferred embodiment: the amount of each of the structures in theLMWH composition, as analyzed by 2D NMR is about that found in Table11A.

In a ninth aspect, the invention features a LMWH composition having oneor more of the following characteristics:

the composition has substantially no (e.g., at least 85%, 90%, 95% ormore of the chains do not have) modified reducing end structures; atleast 60%, 70%, 80%, 85%, 90%, 95%, 99% of the chains of the compositionhave H_(NAc) at the reducing end; less than 90%, 95%, 98%, 99%,preferably none of the chains of the composition have a sulfated ΔU atthe non-reducing end; there is substantially no linkage region (e.g.,less than 0.1% linkage region) present in the composition; thecomposition has more chains with 3-O sulfates than commerciallyavailable LMWHs, e.g., enoxaparin or dalteparin; and the ratio ofΔUH_(NAc,6S)GH_(NS,3S,6S) to ΔU_(2S)H_(NS,6S)IH_(NAc,6S)GH_(NS,3S,6S) inthe composition is about 1:1 to 4:1.

In one embodiment, the composition has two, three, four, five or all ofthese characteristics.

In a tenth aspect, the invention features a LMWH composition having thefollowing structure:

wherein R is H or SO₃X;

R1 is SO₃X or COCH₃;

X is a monovalent or divalent cation;

n=2-50, e.g., 2-40; and

the composition preferably has an average value for n of 9-16, 8-16 or8-15.

In one embodiment, the LMWH composition has the following structure:

wherein:

R is H or SO₃X;

R1 is SO₃X or COCH₃;

X is a monovalent or divalent cation;

n=2-50, e.g., 2-40; and

the composition preferably has an average value for n of 9-16, 8-16 or8-15.

In an eleventh aspect, the invention features, a LMWH composition havingthe following structure:

wherein X is Na or Ca, R is H or SO₃Na;

R1 is SO₃Na or COCH₃;

n=2-45, e.g., 2-35; and

the composition preferably has an average value for n of 7-11 or 8-12.

In one embodiment, the LMWH composition has the following structure:

wherein:

X is Na or Ca, R is H or SO₃Na;

R1 is SO₃Na or COCH₃;

n=2-45, e.g., 2-35; and

the composition preferably has an average value for n of 7-11 or 8-12.

This composition can occur as an intermediate in the production of aLMWH, e.g., as the product of enzymatic digestion of the fast movingfraction (as discussed herein).

In a twelfth aspect, the invention features, a LMWH composition havingthe following structure:

wherein X is Na or Ca, R is H or SO₃Na;

R1 is SO₃Na or COCH₃;

n=2-50, e.g., 2-40; and

the composition preferably has an average value for n of 8 to 15, e.g.,10 to 15, or 9 to 16, e.g., 11 to 16.

In one embodiment, the LMWH composition has the following structure:

wherein X is Na or Ca, R is H or SO₃Na;

R1 is SO₃Na or COCH₃;

n=2-50, e.g., 2-40; and

the composition preferably has an average value for n of 8 to 15, e.g.,10 to 15, or 9 to 16, e.g., 11 to 16.

This composition can occur as an intermediate in the production of aLMWH, e.g., as the product of precipitations to provide a fast movingfraction (as discussed herein).

Any of the LMWHs described herein, e.g., described above, can have otherproperties. E.g., one of the above described compositions can furtherhave one or more of functional or structural properties set out below.

Thus, in one embodiment, the LMWH composition has an anti-Xa activity ofabout 100 to 400 IU/mg, e.g., about 120 to 380 IU/mg, e.g., about 150 to350 IU/mg, e.g., about 170 to 330 IU/mg, e.g., about 180 to 300 IU/mg,e.g., about 150 to 200 IU/mg, 200 to 300 IU/mg, 130 to 220 IU/mg, 225 to274 IU/mg.

In one embodiment, the LMWH composition has an anti-Xa activity that isat least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, 100% neutralizable,e.g., as measured by anti-Xa activity, ACT or aPTT. Preferably, theanti-Xa activity is neutralized by at least 50%, 60%, 70%, 80%, 85%,90%, 95%, 99% or 100% within 5, 10, 15 minutes after protamineadministration. For example, the anti-Xa activity can be neutralized byat least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% within 5, 10,15, 30 minutes after protamine administration at a dose of about 1, 2, 3mg of the LMWH composition per 100 anti-Xa IU of plasma.

In another embodiment, the LMWH composition has one or more of thefollowing properties: the activity of the composition can be monitoredby aPTT and/or ACT; the polydispersity of the composition is less than1.6, e.g., the polydispersity is about 1.6 to 1.1, e.g., 1.5 to 1.1,e.g., 1.4 to 1.1, e.g., 1.3 to 1.1, e.g., 1.2 to 1.1; less than 70%,60%, 50%, 45%, 40%, 35%, 30% of the chains present in the compositionhave a molecular weight greater than 7500 or 8000 Da; less than 40%,35%, 30%, 25% of the chains present in the composition have a molecularweight less than 5500 or 5000 Da; the composition comprises a mixture ofΔU and I/G structures at the non-reducing ends of the chains; and fewerchains in the composition have PF4 binding sites than enoxaparin,dalteparin, UFH.

In one embodiment, about 15%, 20%, 25%, 30%, 35%, 45%, 50% of the chainsin the LMWH composition have a ΔU at the non-reducing end. Preferably,about 15% to 50%, e.g., 15% to 35% of the chains, e.g., 20% to 35% ofthe chains in the composition have a ΔU at the non-reducing end.

In one embodiment, the LMWH composition has a higher degree of sulfationthan enoxaparin or dalteparin. In one embodiment, the LMWH compositionhas more trisulfated disaccharides present in the composition thanenoxaparin or dalteparin, e.g., the LMWH composition has about 50 to 65%trisulfated disaccharides, e.g., 55 to 60%, 55 to 58%, 57 to 60%trisulfated disaccharides, as determined by mole %.

In one embodiment, the composition comprises a higher level ofΔUH_(NAc,6S)GH_(NS,3S,6S) than enoxaparin, daltaparin and/or UFH, e.g.,comprises about 5 to 15 mole %, e.g., 7 to 14 mole %, e.g., 9 to 12 mole%.

In one embodiment, the LMWH composition has a calcium content less than3%, 2.5%, 2%, 1.5%, 1.0%, and/or a sodium content less than 30%, 25%,20%, 15%, 10%. In one embodiment, the LMWH composition comprises: lessthan 1000 ng/mg, 750 ng/mg, 500 ng/mg, 250 ng/mg of a heparinase enzyme,e.g., a heparinase enzyme described herein; less than 1.0%, 0.5%, 0.3%w/w methanol; less than 1.0%, 0.5%, 0.3%, 0.1% w/w ethanol; less than2.0%, 1.75%, 1.25%, 1.0%, 0.5%, 0.3%, 0.15% chloride; less than 15%,10%, 5%, 2.5% water by weight; less than 2000, 1500, 1000, 950, 900,850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300 ppm of freesulfate.

In one embodiment, the LMWH composition provides increased TFPI releaseas compared to enoxaparin. In one embodiment, the LMWH provides at leasta 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 fold increase in TFPIrelease as compared to enoxaparin.

In one embodiment, the LMWH composition has an intravenous half life ofabout 30 minutes to 3 hours, e.g., about 1 to 2 hours. In oneembodiment, the LMWH composition has a subcutaneous half life of about30 minutes to 3.0 or 3.5 hours, e.g., about 1.5 to 2.5 hours, e.g.,about 2 hours.

In one embodiment of any of the first, second, third, fourth, fifth,sixth, seventh, eighth, ninth and tenth aspects, the LMWH compositionhas one or more of the following characteristics:

the composition has substantially no (e.g., at least 85%, 90%, 95% ormore of the chains do not have) modified reducing end structures; atleast 60%, 70%, 80%, 85%, 90%, 95%, 99% of the chains of the compositionhave H_(NAc) at the reducing end; less than 90%, 95%, 98%, 99%,preferably none of the chains of the composition have a sulfated ΔU atthe non-reducing end; there is substantially no linkage region (e.g.,less than 0.1% linkage region) present in the composition; thecomposition has more chains with 3-O sulfates than commerciallyavailable LMWHs, e.g., enoxaparin or dalteparin; and the ratio ofΔUH_(NAc,6S)GH_(NS,3S,6S) to ΔU_(2S)H_(NS,6S)IH_(NAc,6S)GH_(NS,3S,6S) inthe composition is about 1:1 to 4:1 (e.g., 1:1, 2:1, 3:1 or 4:1). In oneembodiment, the LMWH composition has two, three, four, five or all ofthese characteristics.

In another aspect, the invention features, a LMWH composition having thefollowing properties:

-   -   a weight average molecular weight of about 5000 to 9000 Da;    -   anti-IIa activity of about 50 to 300 IU/mg;    -   anti-IIa activity that is at least 50% neutralizable with        protamine, e.g., as measured by ACT or aPTT;    -   ΔUH_(NAc,6S)GH_(NS,3S,6S) is 5 to 15% of the composition,        preferably ΔUH_(NAc,6S)GH_(NS,3S,6S) at the non-reducing end of        about 5 to 15% of the composition;    -   an average chain length of about 9 to 16 disaccharides;    -   an anti Xa to anti-IIa ratio of 3:1 or less;    -   the anti-Xa to anti-IIa ratio remains relatively constant over        the course of an administration of the LMWH, e.g., the anti-Xa        to anti-IIa ratio varies no more than about +1.5, ±1, ±0.5, or        ±0.2, over a period of about 30, 60, 120, 180, 240, 300 minutes.        For example, if an initial ratio of anti-Xa activity to anti-IIa        activity is 2, then the ratio measured at a second time (e.g.,        30, 60, 120, 180, 240, 300 minutes) after the initial        administration will preferably be less than 3, and preferably at        or around 2.

In a preferred embodiment, the LMWH composition has the followingstructure:

wherein:

R is H or SO₃Na;

R1 is SO₃Na or COCH₃;

n=2-50, e.g., 2-40; and

the composition preferably has an average value for n of 9 to 16 or 8 to15.

In a preferred embodiment, the LMWH composition has the followingproperties:

anti-Xa activity of about 100 to 400 IU/mg;

anti-Xa activity that is at least 50% neutralizable, e.g., as measuredby anti-Xa activity, ACT or aPTT;

a polydispersity of less than 1.6;

less than 70%, 60%, 50% of the chains present in the composition have amolecular weight greater than 7500 Da;

less than 40% of the chains present in the composition have a molecularweight less than 5000 Da;

it includes a mixture of ΔU and I/G structures at the non-reducing endsof the chains;

it has substantially no modified reducing end structures;

fewer chains in the composition have PF4 binding sites than enoxaparin,dalteparin, or UFH;

at least 60%, 70%, 80%, 90% of the chains of the composition have HNAcat the reducing end;

about 15% to 35% of the chains in the composition have a ΔU at thenon-reducing end;

less than 90%, 95%, 98%, 99%, preferably none of the chains of thecomposition have a sulfated ΔU at the non-reducing end.

In a preferred embodiment, the LMWH composition has the followingproperties:

it has a higher degree of sulfation than enoxaparin or dalteparin;

it has more trisulfated disaccharides present in the composition thanenoxaparin or dalteparin, e.g., the LMWH composition has about 50 to 65%trisulfated disaccharides, as determined by mole %

it has a higher level of ΔUH_(NAc,6S)GH_(NS,3S,6S) than enoxaparin,daltaparin and/or UFH, e.g., ΔUH_(NAc,6S)GH_(NS,3S,6S) is present atabout 5 to 15 mole %.

In a preferred embodiment, the LMWH composition has the followingproperties:

it has a calcium content less than 3% and/or a sodium content less than20%;

it includes less than 1000 ng/mg of a heparinase enzyme;

it has less than 1.0% w/w methanol;

it has less than 1.0% w/w ethanol;

it has less than 2.0% chloride;

it has less than 15% water by weight;

it has less than 2000 ppm of free sulfate.

In a preferred embodiment, the LMWH composition has the followingproperties:

it provides increased tissue factor pathway inhibitor (TFPI) release ascompared to enoxaparin.

In a preferred embodiment, the LMWH composition has an intravenous halflife of about 30 minutes to 3 hours.

In another aspect, the invention features, a method of making a LMWH.The method includes:

subjecting UFH to one, or a step-wise series, of aqueous alcohol (e.g.,ethanol) precipitations (at least one with a sodium salt (or a saltother than a calcium salt)), to extract a lower molecular weightfraction from the unfractionated heparin (e.g., the fast movingfraction) to provide a first intermediate, wherein the firstintermediate preferably has a average chain length of 10 to 16disaccharides;

digesting the first intermediate using an agent, e.g., an enzyme orchemical, that cleaves glycosidic linkages of unsulfated uronic acids,e.g., an enzyme described herein, e.g., in aqueous buffer, e.g., inaqueous salt buffer, e.g., a sodium acetate buffer, pH of about 5-9,e.g., 7-8, at 25° C. to 52° C., e.g., 37° C., to produce a secondintermediate, wherein the second intermediate preferably has a averagechain length of 8 to 14 disaccharides, e.g., 8-12 disaccharides;

separating high molecular weight high anti-factor Xa and IIa componentsof from the second intermediate from the lower activity materials by asize based step, e.g., size exclusion chromatography (SEC), to producethe third intermediate wherein the third intermediate preferably has aaverage chain length of 9 to 16 disaccharides; and optionally

dissolving the third intermediate in purified water, filtering, e.g.,through a 0.2 pm filter, and lyophilizing to drug substance.

In another aspect, the invention features, a LMWH composition made by amethod described herein.

In another aspect, the method includes an intermediate or reactionmixture from any of the methods for making or analyzing a LMWH describedherein.

In another aspect, the invention features, a pharmaceutical compositionthat includes a LMWH composition described herein.

In one embodiment, the pharmaceutical composition further includes apharmaceutically acceptable carrier.

In one embodiment, the pharmaceutical composition is in a form suitablefor systemic administration. In a preferred embodiment, thepharmaceutical composition is suitable for subcutaneous, intravenous,intra-arterial, intrasynoval, intramuscular, intraperitoneal,intravitreous, epidural, subdural or intrathecal administration. In oneembodiment, a pharmaceutical composition for systemic administration canbe an isotonic solution, e.g., an isotonic solution with or withoutpreservatives. Examples of preservative include, but are not limited to,benzyl alcohol, mannitol and leucine. A unit dosage amount of apharmaceutical composition of the invention can be disposed within apackage or a device suitable for administration. E.g., a compositionsuitable for subcutaneous delivery can be disposed within a syringeconfigured for subcutaneous delivery, a composition suitable forintravenous delivery can be disposed within a syringe configured forintravenous delivery or within another device for intravenous delivery,e.g., an intravenous drip bag or bottle.

In one embodiment, the pharmaceutical composition is in a form suitablefor local invasive administration, e.g., coating or within a devicesuitable for implantation. Examples of devices suitable for implantationinclude, but are not limited to, a stent, and an excorporeal circuit. Inone embodiment, the pharmaceutical composition is in a form suitable forsubcutaneous implantation, implantation into a tissue or organ (e.g., acoronary artery, carotid artery, renal artery, other peripheralarteries, veins, kidney, heart, cornea, vitreous, and cerebrum), orimplantation into a space surrounding a tissue or organ (e.g., kidneycapsule, pericardium, thoracic or peritoneal space.

In one embodiment, the pharmaceutical composition is in a form suitablefor non-invasive administration, e.g., topical, transdermal, pulmonary,nasal, oral, auditory canal, rectal or vaginal administration. A unitdosage amount of a pharmaceutical composition of the invention can bedisposed within a package or a device suitable for such administration.

In one embodiment, the LMWH composition is lyophilized. In anotherembodiment, the LMWH composition is a liquid.

In one embodiment, the invention features a container, e.g., an ampoule,syringe or vial, containing the pharmaceutical composition. In oneembodiment, the LMWH composition is present at about 1500 IU, 2000 IU,2500 IU, 3000 IU, 3500 IU, 4000 IU, 4500 IU, 5000 IU, 5500 IU, 6000 IUanti-Xa activity per mL pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutical composition has an osmolality ofabout 200 to 400 mOsm/L, e.g., about 250 to 350 mOsm/L, about 280 to 330mOsm/L. In one embodiment, the pharmaceutical composition furthercomprises sodium chloride and water.

In another aspect, the invention features, a method of treating asubject including administering a LMWH disclosed herein to the subject.The treatment can be therapeutic, e.g., a treatment which lessens,mitigates or ameliorates an existing unwanted condition or symptomthereof, or prophylactic, e.g., a treatment which delays, e.g.,prevents, the onset of an unwanted condition or symptom thereof. A LMWHcomposition described herein can be used to treat disorders which aretreatable with UFH or with a commercial LMWH, e.g., enoxaparin,daltaparin or tinzaparin. The invention includes methods for treating asubject having, or at risk of having, a disorder or condition selectedfrom the group consisting of: a disorder associated with coagulation,e.g., deep vein thrombosis (DVT) or pulmonary embolism, thrombosis orcardiovascular disease, e.g., acute coronary syndrome (ACS), stable orunstable angina, myocardial infarction, e.g., ST-segment elevatedmyocardial infarction (STEMI) or non-ST-segment elevated myocardialinfarction (NSTEMI), vascular conditions or atrial fibrillation;migraine; atherosclerosis; an inflammatory disorder, such as autoimmunedisease or atopic disorders, psoriasis, arthritis, sepsis; disseminatedintravascular coagulopathy (DIC); an allergy or a respiratory disorder,such as asthma, emphysema, adult respiratory distress syndrome (ARDS),cystic fibrosis, or lung reperfusion injury; stenosis or restenosis; acancer or metastatic disorder; an angiogenic disorder; a fibroticdisorder such as major organ fibrosis, fibroproliferative disorders andscarring associated with trauma; osteoporosis; Alzheimer's; bonefractures such as hip fractures. The subject can be undergoing, or haveundergone, a surgical procedure, e.g., organ transplant, orthopedicsurgery, joint replacement, e.g., hip replacement or knee replacement,percutaneous coronary intervention (PCI), stent placement, angioplasty,or coronary artery bypass graft surgery (CABG). The compositions of theinvention are administered to a subject having or at risk of developingone or more of the diseases in an effective amount for treating thedisorder or condition.

In one embodiment, the method further includes monitoring the activityof the LMWH composition in the subject using a coagulation assay, e.g.,using ACT and/or aPTT.

In one embodiment, the method further includes administering protaminesulfate after administration of the LMWH composition to neutralize someor all of the activity, e.g., anti-Xa activity and/or anti-IIa activity,of the LMWH composition. In one embodiment, about 50%, 60%, 70%, 80%,90%, 95% or all of the anti-IIa activity of the LMWH composition isneutralized, e.g., about 50%, 60%, 70%, 80%, 90%, 95% or all of theanti-IIa activity of the LMWH composition is neutralized within 5, 10,15, 20, 25, 30, 40 minutes after protamine administration. In oneembodiment, about 50%, 60%, 70%, 80%, 90%, 95% or all of the anti-IIaactivity of the LMWH composition is neutralized, e.g., about 50%, 60%,70%, 80%, 90%, 95% or all of the anti-IIa activity of the LMWHcomposition is neutralized within 5, 10, 15, 20, 25, 30, 40 minutesafter protamine administration. In one embodiment, protamine sulfate isadministered at a dose of about 1 mg, 2 mg, 3 mg, 5 mg of the LMWHcomposition per 100 anti-Xa IU of plasma. Neutralization of anti-Xaactivity and/or anti-IIa activity can be determined, e.g., by ACT and/oraPTT.

In another aspect, the invention features, a method of treating (e.g.,therapeutically or prophylactically treating) a disorder, e.g., athrombotic disorder, in a subject. The method includes administering aLMWH composition described herein, to thereby treat, preferably prevent,the disorder. In one embodiment, the disorder is one or more of ACS,myocardial infarction, e.g., NSTEMI OR STEMI, stable angina and unstableangina. Preferably, the thrombotic disorder is arterial thrombosis,e.g., including STEMI. The disorder can be, e.g., associated withsurgical intervention, e.g., PCI, stent placement or angioplasty. Forexample, the subject can have, or be at risk of having, or be recoveringfrom, a surgical intervention, e.g., cardiology intervention (e.g.,angioplasty, PCI, stent placement). In one embodiment, the subject is atrisk for (e.g., is being considered for) receiving surgicalintervention, e.g., CABG.

In one embodiment, the LMWH composition is administered to the subjectintravenously, e.g., at a dose of about 0.03 mg/kg to 0.45 mg/kg, e.g.,0.03 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.22 mg/kg,0.25 mg/kg, 0.27 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.37 mg/kg, 0.4 mg/kg,0.44 mg/kg. In preferred embodiments the LMWH composition isadministered intravenously at a dose of about 0.1 to 0.3 mg/kg, e.g.,0.1 mg/kg, 0.15 mg/kg, 0.20 mg/kg, 0.22 mg/kg, 0.25 mg/kg, 0.27 mg/kg or0.30 mg/kg. In another embodiment, the LMWH composition is administeredto the subject subcutaneously, e.g., at a dose of about 0.1 mg/kg, 0.15mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg, 0.44mg/kg, 0.47 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.60 mg/kg, 0.7 mg/kg, 0.8mg/kg, 0.9 mg/kg, 1.0 mg/kg. In preferred embodiments, the LMWHcomposition is administered subcutaneously at a dose of about 0.15 to1.0 mg/kg, 0.20 to 0.9 mg/kg, 0.25 to 0.9 mg/kg, 0.30 to 0.50 mg/kg,e.g., 0.30 mg/kg, 0.35 mg/kg, 0.40 mg/kg, 0.42 mg/kg, 0.44 mg/kg, 0.47mg/kg or 0.50 mg/kg.

In one embodiment, the method further includes monitoring the activityof the LMWH composition in the subject using a coagulation assay, e.g.,using ACT and/or aPTT. In one embodiment, anti-Xa activity and/oranti-IIa activity is monitored, e.g., with ACT and/or aPTT, prior to,during, or after surgical intervention, e.g., angioplasty, PCI, stentplacement. In one embodiment, anti-Xa activity and/or anti-IIa activityis monitored, e.g., with ACT and/or aPTT, prior to, during, and/or afteradministration of the LMWH composition. In some embodiments, anti-Xaactivity and/or anti-IIa activity is monitored by ACT, and the dose ofLMWH is administered to achieve an ACT of about 200 to 350 seconds.

In one embodiment, the method further includes administering protaminesulfate after administration of the LMWH composition to neutralize someor all of the activity, e.g., anti-Xa activity and/or anti-IIa activity,of the LMWH composition. In one embodiment, at least about 50%, 60%,70%, 80%, 90%, 95% or all of the anti-IIa activity of the LMWHcomposition is neutralized, e.g., at least about 50%, 60%, 70%, 80%,90%, 95% or all of the anti-IIa activity of the LMWH composition isneutralized within 5, 10, 15, 20, 25, 30, 40 minutes after protamineadministration. In one embodiment, at least about 50%, 60%, 70%, 80%,90%, 95% or all of the anti-IIa activity of the LMWH composition isneutralized, e.g., at least about 50%, 60%, 70%, 80%, 90%, 95% or all ofthe anti-IIa activity of the LMWH composition is neutralized within 5,10, 15, 20, 25, 30, 40 minutes after protamine administration. In oneembodiment, protamine sulfate is administered at a dose of about 1-5 mg,e.g., 1 mg, 2 mg, 3 mg, 5 mg of the LMWH composition per 100 anti-Xa IUof plasma. Neutralization of anti-Xa activity and/or anti-Ha activitycan be determined, e.g., by ACT and/or aPTT. In one embodiment, anti-Xaactivity and/or anti-IIa activity can be determined, e.g., by ACT and/oraPTT, prior to, during and/or after administration of protamine sulfate.In one embodiment, anti-Xa activity and/or anti-IIa activity isneutralized prior to, during or after surgical intervention. Forexample, in one embodiment, anti-Xa activity and/or anti-IIa activitycan be neutralized during or after a surgical intervention such asangioplasty or PCI. In another embodiment, the LMWH composition isneutralized prior to surgical intervention such as CABG.

In one embodiment, the method further includes monitoring the patientfor a negative reaction, e.g., epidural or spinal hematoma, hemorrhageor bleeding.

In one embodiment, the LMWH composition is administered intravenously orsubcutaneously.

In one embodiment, the LMWH composition is administered in combinationwith another therapeutic agent, e.g., an anticoagulant or antithromboticagent, e.g., bivalirudin Angiomax), ASA, a GPIIbIIIa inhibitor (e.g.,eptifibatide or abciximab), an ADP inhibitor (e.g., Plavix), rPA,TNKase, aspirin, a P2Y12 inhibitor, a platelet inhibitor, warfarin, andcombinations thereof.

The reversible (neutralizable) and monitorable LMWH compositionsdisclosed herein allow for improved flexibility in treating patients,e.g., patients admitted to the hospital and undergoing evaluation forpossible cardiovascular treatment, e.g., surgery. Accordingly, inanother aspect, the invention features, a method of treating (e.g.,therapeutic or prophylactic treatment) a disorder, e.g., a thrombotic orcardiovascular disorder, in a patient. The method includes:

optionally, administering a reversible and monitorable LMWH compositiondescribed herein to the patient;

classifying the patient (to whom the LMWH composition has been or willbe administered) as not in need of surgical intervention (e.g.,classifying the patient as not in need of surgical intervention prior torelease from the hospital) or as a candidate for surgical interventionprior to release from the hospital;

optionally, if the patient is classified as a candidate for surgicalintervention then performing one or both of monitoring (e.g., asdescribed herein, e.g., with a coagulation assay, e.g., using ACT and/oraPTT) the reversible and monitorable LMWH composition and neutralizing(e.g., as described herein, e.g., by administering protamine sulfate)the reversible and monitorable LMWH composition.

In one embodiment, the LMWH composition is administered to the subjectintravenously, e.g., at a dose of about 0.03 mg/kg to 0.45 mg/kg, e.g.,0.03 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.22 mg/kg,0.25 mg/kg, 0.27 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.37 mg/kg, 0.4 mg/kg,0.44 mg/kg. In preferred embodiments the LMWH composition isadministered intravenously at a dose of about 0.1 to 0.3 mg/kg, e.g.,0.1 mg/kg, 0.15 mg/kg, 0.20 mg/kg, 0.22 mg/kg, 0.25 mg/kg, 0.27 mg/kg or0.30 mg/kg. In another embodiment, the LMWH composition is administeredto the subject subcutaneously, e.g., at a dose of about 0.1 mg/kg, 0.15mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg, 0.44mg/kg, 0.47 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.60 mg/kg, 0.7 mg/kg, 0.8mg/kg, 0.9 mg/kg, 1.0 mg/kg. In preferred embodiments, the LMWHcomposition is administered subcutaneously at a dose of about 0.15 to1.0 mg/kg, 0.20 to 0.8 mg/kg, 0.25 to 0.90 mg/kg, 0.30 to 0.50 mg/kg,e.g., 0.30 mg/kg, 0.35 mg/kg, 0.40 mg/kg, 0.42 mg/kg, 0.44 mg/kg, 0.47mg/kg or 0.50 mg/kg.

In a preferred embodiment, the patient is classified as a candidate forsurgical intervention, e.g., PCI, stent placement or angioplasty, andthe effect of the reversible and monitorable LMWH composition ismonitored. In a preferred embodiment, the surgical intervention isperformed and the reversible and monitorable LMWH composition ismonitored at one or more, or all, of before, during and after thesurgery. In one embodiment, the patient is monitored for an ACT of about200 to 350 before and/or during a surgical intervention such as PCI.

In a preferred embodiment, the patient is classified as a candidate forsurgical intervention, e.g., CABG and the effect of the reversible andmonitorable LMWH composition is one or both of monitored andneutralized. In a preferred embodiment, the surgical intervention isperformed and the reversible and monitorable LMWH composition monitoredone or more, or all of before, during and after the surgery. In oneembodiment, the patient is monitored for an ACT of about 400 to 600,e.g., 400 to 500 prior to surgical intervention such as CABG.

In a preferred embodiment, the subject is being treated for a thromboticdisorder. In one embodiment, the disorder is one or more of ACS,myocardial infarction, e.g., NSTEMI OR STEMI, stable angina and unstableangina. Preferably, the thrombotic disorder is arterial thrombosis,e.g., including ST elevation (STEMI).

In another aspect, the invention features, a method of monitoring asubject treated with a monitorable LMWH composition described herein.The method includes,

optionally, administering a monitorable LMWH composition describedherein to the subject; and

evaluating aPTT and/or ACT in the subject (who has been administered themonitorable LMWH composition).

In one embodiment, a baseline aPTT and/or ACT is determined prior totreating the subject with the LMWH. In one embodiment, the methodincludes comparing aPTT and/or ACT of a subject that has received theLMWH to the baseline aPTT and/or ACT.

In one embodiment, the subject is monitored at one or more, or all, ofthe following stages: prior to, during and after receiving a LMWHcomposition. In one embodiment, the subject is monitored prior to,during and/or after surgical intervention, e.g., PCI, stent placement orangioplasty. In one embodiment, the LMWH composition is monitored for anACT of about 200 to 350 prior to and/or during a surgical interventionsuch as PCI. In another embodiment, the LMWH composition is monitoredfor an ACT of about 400 to 600, e.g., about 400 to 500, prior to CABG.

In another aspect, the invention features, a method of treating asubject who has been administered a reversible LMWH compositiondescribed herein. The method includes:

optionally, administering a reversible LMWH composition described hereinto the subject; and

neutralizing (e.g., as described herein, e.g., by administeringprotamine sulfate) the reversible LMWH composition.

In one embodiment, the subject is monitored at one or more, or all, ofthe following stages: prior to, during and after administration ofprotamine sulfate.

In another aspect, the invention features, a method of advising on, orproviding instructions (e.g., written, oral, or computer generatedinstructions) for, the use of a LMWH having high anti-IIa activity,e.g., a LMWH composition described herein. The method includes providinginstruction regarding use, e.g., with: patients having abnormal renalfunction or diabetes or clot bound thrombin; patients who are candidatesfor PCI, stent placement, CABG, angioplasty, etc.; interventionalcardiology patients; patients in need of neutralization of previouslyadministered LMWH, e.g., neutralizing with protamine sulfate; patientsat risk of epidural or spinal hematoma, hemorrhage and/or bleeding. Inone embodiment, the instruction pertains to administration of the LMWHcomposition for ACS, myocardial infarction, e.g., NSTEMI OR STEMI,stable angina and unstable angina, e.g., administration in a subpopulation of patients such as patients having abnormal renal function,or elderly patients (e.g., patients over 60 years of age). In oneembodiment, the instruction pertains to administration of the LMWHcomposition for thrombotic disorders, e.g., thrombotic disordersassociated with surgical intervention, e.g., PCI, stent replacement orangioplasty.

In another aspect, the invention features, a method of advising on theuse of a LMWH composition described herein, that includes providinginstruction regarding monitoring anti-Xa activity and/or anti-IIaactivity using ACT and/or aPTT.

In another aspect, the invention features, a method of manufacturing aLMWH composition, e.g., a LMWH composition described herein. The methodincludes one or more of the following steps:

(1) subjecting a glycosaminoglycan (GAG) containing sample, e.g., UFH,to a first a precipitation, e.g., with a polar organic solvent (e.g., analcohol, e.g., ethanol), a polar non-organic solvent (e.g., water), anda salt (e.g., a sodium salt, e.g., sodium acetate, or calcium salt,e.g., calcium acetate), to yield a first supernatant;

(2) subjecting the first supernatant to a second precipitation, e.g.,with a polar organic solvent (e.g., an alcohol, e.g., ethanol), and apolar non-organic solvent (e.g., water), to yield a precipitate (thiscan be used to provide a fast moving fraction as discussed elsewhereherein);

(3) solubilizing the precipitate, preferably in water, and cleaving thesolubilized precipitate with an agent that cleaves glycosidic linkagesof unsulfated uronic acids, e.g., adjacent to an N-acetyl glucosamineresidue. An example is a heparinase III enzyme described herein,preferably MO11, preferably in the presence of sodium acetate, andpreferably to completion, e.g., as indicated by a UV plateau, to providea cleaved preparation;

(4) precipitating the cleaved preparation, e.g., with a salt, e.g., asodium salt, preferably sodium chloride, and a polar organic solvent,e.g., an alcohol, e.g., methanol, to form solids, having saccharideswith e.g., an average chain length of 8-14, e.g., 8-12 disaccharides;

(5) subjecting material from the solids to a purification step, e.g., achromatographic purification step, e.g., size selection step, e.g.,exclusion chromatography, ion exchange chromatography, or filtration, toprovide a preparation with a higher average molecular weight than instep (4). In a preferred embodiment the higher average molecular weightpreparation has an average chain length of 9 to 16 disaccharides or anaverage molecular weight of 5000 to 9000 Da.

In another aspect, the invention features, methods of making a LMWHcomposition having an average chain length of about 9 to 16disaccharides. The method includes:

providing a precursor LMWH composition (e.g., a intermediate compositionfrom a method described herein) having an average chain length of lessthan 9 to 16 disaccharides, preferably about 8 to 14 disaccharides,e.g., 8 to 12 disaccharides; and

processing the precursor LMWH composition to obtain a LMWH having anaverage chain length of about 9 to 16 disaccharides.

Preferably, the processing includes size-based selection, e.g., asize-dependent separation, e.g., by one or more of size exclusionchromatography, ion exchange chromatography or filtration.

In one embodiment, the precursor LMWH composition is a preparationhaving an average chain length of about 8 to 14 disaccharides, e.g., 8to 12 disaccharides. In a preferred embodiment it was obtained by amethod including salt precipitation and enzymatic digestion of a highermolecular weight preparation, e.g., UFH. In one embodiment, the salt isa salt of a monovalent or divalent cation. Examples of monovalent anddivalent cations that can be used include, e.g., sodium, potassium,rubidium, cesium, barium, calcium, magnesium, strontium, andcombinations thereof. In one embodiment, the salt of monovalent ordivalent cation is an acetate of a monovalent or divalent cation.

In one embodiment, the enzyme (or enzymes) used for digestion cleaves atone or more glycosidic linkages of unsulfated uronic acids, e.g.,adjacent to an N-acetyl glucosamine residue. Examples of enzymes thatcan be used include, e.g., heparinase III, mutants of heparinase III,e.g., a heparinase III mutant described in U.S. Pat. No. 5,896,789(e.g., a mutant of heparinase III having one or more histidine residueselected from the group consisting of His 36, His105, His110, His139,His152, His225, His234, His 241, His424, His469, and His539 has beensubstituted with an alanine), and heparin sulfate glycosaminoglycanlyase III from Bacteroides thetaiotaomicron. In a preferred embodiment,the enzyme used for digestion is a mutated heparinase III having analanine at residue 225 of the amino acid sequence substituted with analanine.

In a preferred embodiment, the precursor composition can be obtained by:

(1) subjecting a glycosaminoglycan (GAG) containing sample, e.g., UFH,to a first a precipitation, e.g., with a polar organic solvent (e.g., analcohol, e.g., ethanol), a polar non-organic solvent (e.g., water), anda salt (e.g., a sodium salt, e.g., sodium acetate, or a calcium salt,e.g., calcium acetate), to yield a first supernatant;

(2) subjecting the first supernatant to a second precipitation, e.g.,with a polar organic solvent (e.g., an alcohol, e.g., ethanol), and apolar non-organic solvent (e.g., water), to yield a precipitate (thiscan be used to provide a fast moving fraction as discussed elsewhereherein);

(3) solubilizing the precipitate and cleaving the solubilizedprecipitate with a heparinase III enzyme, preferably MO11, preferably inthe presence of sodium acetate, and preferably to completion as, e.g.,indicated by UV absorption of greater than 9.8, to provide a cleavedpreparation.

In one aspect, the invention features a method of making a LMWHcomposition, e.g., a LMWH composition described herein. The methodincludes:

(1) subjecting a glycosaminoglycan (GAG) containing sample, e.g., UFH,to a first a precipitation, e.g., with a polar organic solvent (e.g., analcohol, e.g., ethanol), a polar non-organic solvent (e.g., water), anda sodium salt (e.g., sodium acetate), to yield a first supernatant;

(2) subjecting the first supernatant to a second precipitation, e.g.,with a polar organic solvent (e.g., an alcohol, e.g., ethanol), and apolar non-organic solvent (e.g., water), to yield a precipitate (thiscan be used to provide a fast moving fraction as discussed elsewhereherein);

(3) optionally solubilizing the precipitate and cleaving the solubilizedprecipitate with a enzyme described herein, preferably in the presenceof sodium acetate, and preferably to completion as, e.g., indicated byUV absorption of greater than 9.8, to provide a cleaved preparation; and

(4) optionally processing the fraction to produce a LMWH preparation.

In one embodiment, the enzyme (or enzymes) used for digestion cleaves atone or more glycosidic linkages of unsulfated uronic acids, e.g.,adjacent to an N-acetyl glucosamine residue. Examples of enzymes thatcan be used include, e.g., heparinase III, mutants of heparinase III,e.g., a heparinase III mutant described in U.S. Pat. No. 5,896,789(e.g., a mutant of heparinase III having one or more histidine residueselected from the group consisting of His 36, His105, His110, His139,His152, His225, His234, His 241, His424, His469, and His539 has beensubstituted with an alanine), and heparin sulfate glycosaminoglycanlyase III from Bacteroides thetaiotaomicron. In a preferred embodiment,the enzyme used for digestion is a mutated heparinase III having analanine at residue 225 of the amino acid sequence substituted with analanine.

In one embodiment, the digested fraction is the final product. In otherembodiments, the method can include one or more additional processingsteps to obtain a final product. In one embodiment, the method includesprocessing the digested fraction to obtain a LMWH composition having anaverage chain length of 9 to 16 disaccharides.

In one embodiment, size exclusion chromatography, ion exchangechromatography and/or filtration can be used to obtain a LMWHcomposition having an average chain length of 9 to 16 disaccharides.

In another aspect, the invention features, methods of evaluating orprocessing a GAG such as UFH to determine suitability of the GAG forprocessing into a LMWH composition, e.g., a LMWH composition describedherein. The method includes determining the quantity of N-acetyl presentin a GAG preparation, comparing the quantity to a preselected criterionand making a decision about the GAG preparation based upon the whetherthe preselected criterion is met. In a preferred embodiment, a decisionor step is taken, e.g., the GAG preparation is classified, accepted ordiscarded, processed into a drug substance or drug product, or a recordmade or altered to reflect the determination, depending upon whether thepreselected criterion is met. In some embodiments, when the preselectedcriterion is not met, a decision can be made about altering one or moresteps in manufacturing of a LMWH composition.

In one embodiment, the preselected criterion is N-acetyl present in theGAG preparation at an amount of about 11% or higher, e.g., as determinedby mole %, relative to total glucosamine content. A GAG preparationhaving N-acetyl content within this range is indicative of a GAGpreparation suitable for processing into a LMWH composition, e.g., aLMWH composition described herein. In such embodiments, when thispreselected criterion is met, the GAG preparation is accepted andprocessed into intermediates, drug substance or drug product.

In one embodiment, the amount of N-acetyl present in a GAG preparationcan be determined using, e.g., nuclear magnetic resonance (NMR).

In preferred embodiments, methods disclosed herein are useful from aprocess standpoint, e.g., to monitor or ensure batch-to-batchconsistency or quality, or to evaluate a sample with regard to apreselected criterion.

In one aspect, the invention features, a method of evaluating orprocessing an intermediate LMWH preparation, e.g., produced by a methoddescribed herein, to determine suitability of the intermediatepreparation for processing into a LMWH composition. The intermediateLMWH preparation is a fast moving fraction obtained, e.g., by saltprecipitation with sodium or sodium acetate of a glycosaminoglycan (GAG)containing sample in a solvent as described herein. The method includescomparing the quantity of one or more of structural moieties, e.g., oneor more of sulfated iduronic acid, N-sulfated hexosamine linked touronic acid, epoxide and 6-O sulfated hexosamine, in the intermediateLMWH preparation to the quantity of the same structural moiety inunfractionated heparin starting material, and making a decision aboutthe intermediate LMWH preparation based upon whether a preselectedcriterion between the starting material and intermediate LMWHpreparation is met. In a preferred embodiment, a decision or step istaken, e.g., the intermediate LMWH preparation is classified, acceptedor discarded, processed into a drug substance or drug product, or arecord made or altered to reflect the determination, depending uponwhether the a preselected relationship is met. In some embodiments, whenthe preselected criterion is not met, a decision can be made aboutaltering one or more steps in manufacturing of a LMWH composition.

In one embodiment, the preselected criterion is a decrease in sulfatediduronic acid in the intermediate preparation as compared to thestarting material. An intermediate preparation having a decreasedsulfated iduronic acid content is indicative of an intermediatepreparation suitable for further processing into a LMWH composition,e.g., a LMWH composition described herein. In such embodiments, whenthis preselected criterion is met, the intermediate preparation isaccepted and processed into further intermediates, drug substance ordrug product.

In one embodiment, the preselected criterion is an increase inN-sulfated hexosamine linked to uronic acid (e.g., iduronic and/orglucuronic acid) in the intermediate preparation as compared to thestarting material. An intermediate preparation having an increasedN-sulfated hexosamine linked to uronic acid is indicative of anintermediate preparation suitable for further processing into a LMWHcomposition, e.g., a LMWH composition described herein. In suchembodiments, when this preselected criterion is met, the intermediatepreparation is accepted and processed into further intermediates, drugsubstance or drug product.

In one embodiment, the preselected criterion is a decrease in epoxide inthe intermediate preparation as compared to the starting material. Anintermediate preparation having a decreased epoxide content isindicative of an intermediate preparation suitable for furtherprocessing into a LMWH composition, e.g., a LMWH composition describedherein. In such embodiments, when this preselected criterion is met, theintermediate preparation is accepted and processed into furtherintermediates, drug substance or drug product.

In one embodiment, the preselected criterion is an increase in 6-Osulfated hexosamine in the intermediate preparation as compared to thestarting material. An intermediate preparation having increased 6-Osulfated hexosamine is indicative of an intermediate preparationsuitable for further processing into a LMWH composition, e.g., a LMWHcomposition described herein. In such embodiments, when this preselectedcriterion is met, the intermediate preparation is accepted and processedinto further intermediates, drug substance or drug product.

In one embodiment, the amount of a structural moiety in the startingmaterial and/or intermediate preparation is determined using one or moreof nuclear magnetic resonance (NMR), capillary electrophoresis (CE) andhigh performance liquid chromatography (HPLC).

In preferred embodiments, methods disclosed herein are useful from aprocess standpoint, e.g., to monitor or ensure batch-to-batchconsistency or quality, or to evaluate a sample with regard to apreselected criterion.

Certain characteristics can make a UFH sample a more preferred startingmaterial for making a LMWH of the inventions. Accordingly, in anotheraspect, the invention provides a method of evaluating a UFH preparationas a starting material to make a LMWH composition described herein.

The method includes providing an evaluation of the UFH preparation for aparameter related to suitability of the UFH sample for use in the makingof a LMWH described herein; and optionally, providing a determination ofwhether a value (e.g., a value correlated to presence, amount,distribution, or absence) determined for the parameter meets apreselected criterion, e.g., is present, or is present within apreselected range, thereby evaluating the UFH sample.

In a preferred embodiment, the criterion is satisfied and the UFH sampleis selected and processed into the LMWH.

In a preferred embodiment, the parameter is the presence or amount of astructure listed in Table 2, preferably one related to the efficacy of astep in the method of making the LMWH, e.g., a structure which promotesor is positively correlated with cleavage by a heparinase, e.g.,H_(NAc(internal)).

In a preferred embodiment, the method includes determining if the amountof H_(NAc(internal)) in the UFH sample has a predetermined relationshipwith a reference, e.g., it is equal to or greater than a preselectedreference value.

In a preferred embodiment, a value for the parameter in an intermediateused in making the LMWH is also determined and optionally, that valuemust also meet a predetermined criterion to select the UFH for use inmaking the LMWH.

In one aspect, the invention provides a method of evaluating a UFHpreparation, as a starting material to make a LMWH composition describedherein.

The method includes optionally, performing an operation, e.g., aprecipitation, on the UFH sample to provide an intermediate (preferablythe steps used to produce this intermediate and the intermediate are thesame as the steps and an intermediate of the method used to make theLMWH); providing an evaluation of the intermediate preparation for aparameter related to suitability of the UFH sample for use in the makingof a LMWH described herein; and optionally, providing a determination ofwhether a value (e.g., a value correlated to presence, amount,distribution, or absence) determined for the parameter meets apreselected criterion, e.g., is present, or is present within apreselected range, thereby evaluating the UFH preparation.

In a preferred embodiment, the criterion is satisfied and the UFH sampleis selected and processed into the LMWH.

In a preferred embodiment, the parameter is the presence or amount of astructure listed in Table 2, preferably one related to the efficacy of astep in the method of making the LMWH, e.g., a structure which promotesor is positively correlated with cleavage by a heparinase, e.g.,H_(NAc(internal)).

In a preferred embodiment, the method includes determining if the amountof H_(NA(internal)) in the intermediate sample has a predeterminedrelationship with a reference, e.g., it is equal to or greater than apreselected reference.

In a preferred embodiment, a value for the parameter in the UFH is alsodetermined and optionally, that value must also meet a predeterminedcriterion to select the UFH for use in making the LMWH.

In preferred embodiments of either of these methods, a decision or stepis taken, e.g., the sample is classified, selected, accepted ordiscarded, released or withheld, processed into a drug product, shipped,moved to a different location, formulated, labeled, packaged, releasedinto commerce, or sold or offered for sale, or a record made or alteredto reflect the determination, depending on whether the preselectedcriterion is met. E.g., based on the result of the determination orwhether one or more subject entities is present, or upon comparison to areference standard, the batch from which the sample is taken can beprocessed, e.g., as just described.

In either method, a preferred embodiment includes analyzing the samplewith NMR.

In a preferred embodiment, either method can include providing acomparison of the value determined for a parameter with a referencevalue or values, to thereby evaluate the sample. In preferredembodiments, the comparison includes determining if the test value has apreselected relationship with the reference value, e.g., determining ifit meets the reference value. The value need not be a numerical valuebut, e.g., can be merely an indication of whether the subject entity ispresent.

A preferred embodiment of either method can include determining if atest value is equal to or greater than a reference value, if it is lessthan or equal to a reference value, or if it falls within a range(either inclusive or exclusive of one or both endpoints).

In preferred embodiments of either method, the test value, or anindication of whether the preselected criterion is met, can bememorialized, e.g., in a computer readable record.

In preferred embodiments of either method, the intermediate is preparedby one or more or all of the following steps:

(1) subjecting a UFH sample to a first a precipitation, e.g., with apolar organic solvent (e.g., an alcohol, e.g., ethanol), a polarnon-organic solvent (e.g., water), and a salt (e.g., a sodium salt,e.g., sodium acetate), to yield a first supernatant;

(2) subjecting the first supernatant to a second precipitation, e.g.,with a polar organic solvent (e.g., an alcohol, e.g., ethanol), and apolar non-organic solvent (e.g., water), to yield a precipitate (thiscan be used to provide a fast moving fraction as discussed elsewhereherein);

(3a) solubilizing the precipitate, preferably in water;

(3b) cleaving the solubilized precipitate with an enzyme (or enzymes)that cleaves glycosidic linkages of unsulfated uronic acid, e.g.,adjacent to an N-acetyl glucosamine residue, e.g., heparinase IIIenzyme, preferably MO11, preferably in the presence of sodium acetate,and preferably to completion as, e.g., indicated by UV absorption ofgreater than 9.8, to provide a cleaved preparation

The preferred intermediate is that produced in step (2) or (3a), thoughthe method can use others.

In another aspect, the invention features, a method of evaluating a LMWHpreparation described herein. The method includes: providing a LMWHpreparation described herein; determining if a structure, activity orfunction described herein is present in or possessed by the preparation,thereby evaluating a LMWH preparation described herein. In a preferredembodiment, the determining includes determining if the structure,activity or function is present at a preselected level or in apreselected range, e.g., a level or range disclosed herein.

Accordingly, in one aspect, the invention provides a method ofevaluating or processing a LMWH composition described herein. The methodincludes: providing an evaluation of a parameter related to a peaklisted in Table 10A. Such parameters can include, or be a function of,the presence, relative distribution, or amount of a peak, and,optionally, providing a determination of whether a value (e.g., a valuecorrelated to presence, amount, distribution, or absence) determined forthe parameter meets a preselected criterion, e.g., is present, or ispresent within a preselected range, thereby evaluating or processing themixture.

In a preferred embodiment, the method includes analyzing, e.g.,separating, a digest of the sample by digestion with heparinase I,heparinase II, heparinase III by electrophoresis, e.g., capillaryelectrophoresis.

In a preferred embodiment, the method includes evaluating a sample todetermine if one or more of the peaks listed in Table 10A is present.

In a preferred embodiment, the method includes providing a comparison ofthe value determined for a parameter with a reference value or values,to thereby evaluate the sample. In preferred embodiments, the comparisonincludes determining if the test value has a preselected relationshipwith the reference value, e.g., determining if it meets the referencevalue. The value need not be a numerical value but, e.g., can be merelyan indication of whether the subject entity is present.

In a preferred embodiment, the method includes determining if a testvalue is equal to or greater than a reference value, if it is less thanor equal to a reference value, or if it falls within a range (eitherinclusive or exclusive of one or both endpoints). By way of example, theamount of a peak listed in Table 10A can be determined and, optionallyshown to fall within a preselected range, e.g., a range whichcorresponds to a range from Table 10A. In a preferred embodiment: theamount of each peak is about that found in Table 10A, the amount of eachpeak is within a range provided in Table 10A; the amount of peaks 10 and11 are with a range provided in Table 10A.

In preferred embodiments, the test value, or an indication of whetherthe preselected criterion is met, can be memorialized, e.g., in acomputer readable record.

In preferred embodiments, a decision or step is taken, e.g., the sampleis classified, selected, accepted or discarded, released or withheld,processed into a drug product, shipped, moved to a different location,formulated, labeled, packaged, released into commerce, or sold oroffered for sale, or a record made or altered to reflect thedetermination, depending on whether the preselected criterion is met.E.g., based on the result of the determination or whether one or moresubject entities is present, or upon comparison to a reference standard,the batch from which the sample is taken can be processed, e.g., as justdescribed.

The structures in Table 10A can be determined using CE, and whennecessary, by other analytical methods.

In another aspect, the invention features, a method of evaluating orprocessing a LMWH preparation described herein. The method includes:

providing a LMWH preparation which has been digested with heparinase I,heparinase II, heparinase III and separated by a separation techniquesuch as CE;

determining if one or more of the peaks listed in Table 10A is present.

In a preferred embodiment, the method includes determining if a peaklisted in Table 10A falls within a preselected range from Table 10A. Ina preferred embodiment: the amount of each peak is about that found inTable 10A, the amount of each peak is within a range provided in Table10A; the amount of peaks 10 and 11 are with a range provided in Table10A.

In another aspect, the invention provides a method of evaluating orprocessing a LMWH composition described herein.

The method includes providing an evaluation of a parameter related tothe structure or structures of Table 11A. Such parameters can include,or be a function of, the presence, relative distribution, or amount of astructure, and, optionally, providing a determination of whether a value(e.g., a value correlated to presence, amount, distribution, or absence)determined for the parameter meets a preselected criterion, e.g., ispresent, or is present within a preselected range, thereby evaluating orprocessing the mixture.

In a preferred embodiment, the method includes analyzing the compositionusing 2D-NMR.

In a preferred embodiment, the method includes evaluating a sample todetermine if one or more of the structures provided in Table 11A ispresent.

In a preferred embodiment, the method includes providing a comparison ofthe value determined for a parameter with a reference value or values,to thereby evaluate the sample. In preferred embodiments, the comparisonincludes determining if the test value has a preselected relationshipwith the reference value, e.g., determining if it meets the referencevalue. The value need not be a numerical value but, e.g., can be merelyan indication of whether the structure is present.

In a preferred embodiment, the method includes determining if a testvalue is equal to or greater than a reference value, if it is less thanor equal to a reference value, or if it falls within a range (eitherinclusive or exclusive of one or both endpoints). By way of example, theamount of a structure provided in Table 11A can be determined and,optionally shown to fall within a preselected range, e.g., a range whichcorresponds to a range from Table 11A. In a preferred embodiment: theamount of each structure is about that found in Table 11A, the amount ofeach structure is within a range provided in Table 11A.

In preferred embodiments, the test value, or an indication of whetherthe preselected criterion is met, can be memorialized, e.g., in acomputer readable record.

In preferred embodiments, a decision or step is taken, e.g., the sampleis classified, selected, accepted or discarded, released or withheld,processed into a drug product, shipped, moved to a different location,formulated, labeled, packaged, released into commerce, or sold oroffered for sale, or a record made or altered to reflect thedetermination, depending on whether the preselected criterion is met.E.g., based on the result of the determination or whether one or morestructures is present, or upon comparison to a reference standard, thebatch from which the sample is taken can be processed, e.g., as justdescribed.

The structures in Table 11A can be determined using 2D NMR, and whennecessary, by other analytical methods.

In another aspect, the invention features, a method of evaluating orprocessing a LMWH preparation described herein. The method includesproviding a LMWH preparation which has been analyzed using 2D NMR;determining if one or more of the structure listed in Table 11A ispresent.

In a preferred embodiment, the method includes determining if astructure listed in Table 11A falls within a preselected range, e.g., arange which corresponds to a range 1 from Table 11A. In a preferredembodiment: the amount of each structure is about that found in Table11A, the amount of each structure is within a range provided in Table11A.

Some methods described herein include making a determination of whethera subject entity is present at a preselected level or within apreselected range and that level or range is expressed in specific unitsof measurement, e.g., mole %, e.g., present in a range of X-Y mole %.One can perform the method by determining the amount of subject entityin terms of mole % and then compare that with a reference expressed inmole %, in this example, X-Ymole %. One need not, however, make themeasurement in terms of mole % and compare it with reference valuesexpressed in mole %. The sample has an actual level of subject entity,which can be expressed as X-Y when described in units of mole %. Thatactual level can also be expressed in other units, e.g., weight %. Thatactual level is the same regardless of the units in which it isexpressed. The specification of mole % in the method is merely toindicate the actual prevalence of the subject entity. The level ofsubject entity can be measured in terms of other units and the referencevalue can be expressed in terms of other units, as long as the referencevalue as expressed in terms of alternative units corresponds to the sameamount of subject entity as the reference value expressed in mole %,e.g., X-Ymole % in this example. Thus, a method which requires showingthe subject entity is present at X-Y mole % can be performed by showingthat the subject entity is present in a range expressed in analternative unit of measure, e.g., weight %, chain number, or % AUC,wherein the range, as described in the alternative unit of measure,corresponds to the same amount of subject entity which would give themole % referred to, in this example X-Y mole %.

One can establish a functionally equivalent range for an alternativeunit of measure by applying art known methods in conjunction with thisspecification. E.g., one can provide samples in the range of X-Y mole %,and then establish the corresponding range for those samples for interms of an alternative unit of measure.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF THE DRAWINGS

The drawings are first briefly described.

FIG. 1 is a flow chart depicting the four steps of manufacturing processof M118-REH.

FIG. 2A is a graph depicting capillary electrophoresis profile ofenoxaparin digested with heparinase I, heparinase II and heparinase III.FIG. 2B is a graph depicting capillary electrophoresis profile ofM118-REH digested with heparinase I, heparinase II and heparinase III.

FIG. 3 is a graph depicting a comparison of the UV and fluorescenceprofiles generated by post-column labeling for a digest of M118-REHanalyzed by Ion-pairing RP HPLC. The top trace reflects UV 232 nmdetection, and the bottom trace reflects fluorescence detection at 410nm. The species labeled by arrows show up mainly in the fluorescenceprofile and are not observed in the UV profile; they representnon-reducing end saccharides of M118-REH chains that arise from thestarting UFH.

FIG. 4 is graph depicting a two-dimensional NMR HSQC analysis ofM118-REH.

FIG. 5 is a diagram depicting formation of reducing and non-reducingends.

FIG. 6 is a manufacturing process flow diagram of M118-REH Injectable.

FIG. 7 is a graph depicting in vitro neutralization of LMWHs byprotamine sulfate. “M118” (shorthand in this figure for M118-REH) isrepresented by a lighter dot; enoxaparin sodium is represented by adarker dot. Graph was plotted with percentage of remaining anti-Xaactivity against the ratio of protamine to LMWHs activity.

FIG. 8 is a graph depicting TFPI release from human umbilical veinendothelial cells (HUVEC) by different heparins at 0.01 mg/ml (wellsn=3, mean±STDEM). There is a statistically significance between M118(which is shorthand for M118-REH in this figure) (second bar from theleft in each group) and control (first bar from the left in each group)and control groups (p<0.01) after both 24 and 48 hours incubation.

FIG. 9 is a graph depicting the pharmacodynamics of M118-REH bymeasuring ACT and aPTT after intravenous injection in the NHP model.

FIG. 10 is a graph depicting comparison of TTO of M118-REH 0.5 (thirdcolumn) and 1 mg/kg (sixth column) with enoxaparin sodium 2, 3 and 4mg/kg (second, fourth, and fifth, respectively) intravenously injectedin ferric chloride induced thrombosis model. All treatment groups havesignificant longer TTO compared with Control (first column). There is astatistical significant difference between M118-REH (1 mg/kg) andenoxaparin sodium 3 mg/kg (p<0.01).

FIG. 11 is a graph depicting neutralization of anti-Xa activity ofheparins in Sprague-Dawley rat model (M118-REH, enoxaparin sodium andUFH) by protamine sulfate. Graph was plotted with percent of remaininganti-Xa activity vs. time at ratios of 0.5 or 1 mg:100 anti-Xa IU ofprotamine to heparin (or 0.5 or 1 mg: 1 mg in case of UFH).

FIG. 12 is a graph depicting correlation of ACT measurement and anti-Xaactivity of M118-REH (triangle and line), enoxaparin (open circle withdashed line) and UFH (square with dashed line). This data suggests thatthe M118-REH at the specified doses demonstrates the best correlation ofACT with anti-Xa activity when compared to enoxaparin or UFH (r²=0.85).

FIG. 13. Anti-Factor Xa activity (top) and anti-Factor IIa activity(bottom) vs. time in a canine model of deep arterial thrombosis(Lucchesi's model). The vehicle control group shown in the graphssubsequently received M118-REH at 150 IU/kg. Error bars are ±SE. UFH,unfractionated heparin.

FIG. 14. Correlation of anti-Factor Xa and IIa activities in a caninemodel of deep arterial thrombosis (Lucchesi's model). Individual pointsrepresent data from a single animal. All animals in all treatment groupsare shown. Correlation coefficients (r²) were 0.890 and 0.465 in theM118-REH and unfractionated heparin (UFH) groups, respectively.

FIG. 15. Anti-Factor Xa:IIa ratio over time in a canine model of deeparterial thrombosis. Error bars are +SE (top halves only are shown tomaximize clarity). UFH, unfractionated heparin.

FIG. 16. Coagulation activity vs. time as assessed by ACT (top), aPTT(middle), and PT (bottom) assays in a canine model of deep arterialthrombosis. The vehicle control group shown in the graphs subsequentlyreceived M118-REH at 150 IU/kg. Error bars are ±SE. UFH, unfractionatedheparin.

FIG. 17. Percentage of animals with occluded femoral arteries in acanine model of deep arterial thrombosis. Animals were monitored byDoppler flow for up to 180 minutes post current initiation. Occlusionwas defined as blood flow through an injured artery that was ≦2% ofbaseline flow. UFH, unfractionated heparin.

DETAILED DESCRIPTION Optimized LMWHs

In many clinical settings, commercially available LMWH preparations arepreferred over UFH preparations because LMWHs have more predictablepharmacokinetics and can be administered subcutaneously. However,currently available LMWH preparations lack many of the desirableproperties of UFH such as substantial anti-IIa activity, reversibility(or neutralizability) with protamine sulfate and monitorability. Thus,there are clinical settings where LMWHs are not an optimal or practicaltreatment choice. The invention features LMWH preparations designed tohave properties that are clinically advantageous, e.g., over othercommercially available LMWH preparations and UFH preparations. Suchproperties include, e.g., one or more of: reversibility with proteominesulfate; predictable pharmacokinetics, anti-IIa activity; substantiallyconstant anti-Xa activity to anti-IIa activity ratio; monitorableactivity levels by standard tests such as, e.g., ACT or aPTT;subcutaneous bioavailability; and reduced occurrence of HIT.

Anti-IIa Activity

LMWH preparations are disclosed herein that include a significant numberof chains of sufficient length (which can be described, e.g., in termsof average chain length of the preparation and/or weight averagemolecular weight of the preparation) to provide anti-IIa activity, e.g.,anti-IIa activity of about 50 to 300 IU/mg, about 70 to 280 IU/mg, about90 to 250 IU/mg, about 100 to 140 IU/mg, about 100 to 140 IU/mg, about150 to about 200 IU/mg, about 130 to 190 IU/mg, about 155 to 195 IU/mg.Anti-IIa activity is calculated in International Units of anti-IIaactivity per milligram using the statistical methods for parallel lineassays. The anti-IIa activity levels described herein are measured usingthe following principle.

M118+ATIII→[M118·ATIII]

IIa

M118→ATIII[M118·ATIII·IIa]+IIa (Excess)

IIa (Excess)+Substrate→Peptide+pNA (measured spectrophotometrically)

Anti-factor IIa activity is determined by the sample potentiating effecton antithrombin (ATIII) in the inhibition of thrombin. Thrombin excesscan be indirectly spectrophotometrically measured. The anti-factor IIaactivity can be measured, e.g., on a Diagnostica Stago analyzer or on anACL Futura3 Coagulation system, with reagents from Chromogenix (S-2238substrate, Thrombin (53 nkat/vial), and Antithrombin), or on anyequivalent system. Analyzer response is calibrated using the 2ndInternational Standard for Low Molecular Weight Heparin.

Chain Length/Molecular Weight

A determination of whether a LMWH preparation includes chains ofsufficient chain length can be made, for example, by determining theaverage chain length of the chains in the LMWH preparation and/or bydetermining the weight average molecular weight of chains within theLMWH preparation. When average chain length is determined, an averagechain length of about 5 to 20, e.g., 7 to 18, preferably about 9 to 16or 8 to 14 disaccharide repeats, indicates that a significant number ofchains in the LMWH preparation are of sufficient chain length.

“Average chain length” as used herein refers to the average chain lengthof uronic acid/hexosamine disaccharide repeats that occur within achain. The presence of non-uronic acid and/or non-hexosamine buildingblocks (e.g., attached PEG moieties) are not included in determining theaverage chain length. Average chain length is determined by dividing thenumber average molecular weight (Mn) by the number average molecularweight for a disaccharide (500 Da). Methods of determining numberaverage molecular weight are described below using SEC MALS.

Examples of such LMWH preparations include the following:

wherein R is H or SO₃X;R1 is SO₃X or COCH₃ and X is a monovalent or divalent cation (e.g., Naor Ca);and average n is about 9 to 16 or 8 to 15;

wherein

R is H or SO₃X;

R1 is SO₃X or COCH₃, X is a monovalent or divalent cation (e.g., Na orCa);and average n is about 9 to 16 or 8 to 15;

wherein,

X is a monovalent or divalent cation (e.g., Na or Ca);

R is H or SO₃X;

R1 is SO₃X or COCH₃; and

average n is about 8 to 12 or 7 to 11; and

wherein,

X is a monovalent or divalent cation (e.g., Na or Ca);

R is H or SO₃X;

R1 is SO₃X or COCH₃;

average n is 8 to 12 or 7 to 11, and

When weight average molecular weight of a preparation is determined, aweight average molecular weight of about 5000 to 9000 Da, about 5000 to8300 Da, preferably about 5500 to 8000 Da, about 5700 to 7900, or about5800 to 6800 Da, indicates that a significant number of chains in theLMWH preparation are of sufficient chain length.

“Weight average molecular weight” as used herein refers to the weightaverage in daltons of chains of uronic acid/hexosamine disacchariderepeats. The presence of non-uronic acid and/or non-hexosamine buildingblocks are not included in determining the weight average molecularweight. Thus, the molecular weight of non-uronic acid and non-hexosaminebuilding blocks within a chain or chains in the preparation should notbe included in determining the weight average molecular weight. Theweight average molecular weight (M_(w)) is calculated from the followingequation: M_(w)=Σ(c_(i)m_(i))/Σc_(i). The variable ci is theconcentration of the polymer in slice i and Mi is the molecular weightof the polymer in slice i. The summations are taken over achromatographic peak, which contains many slices of data. A slice ofdata can be pictured as a vertical line on a plot of chromatographicpeak versus time. The elution peak can therefore be divided into manyslices. The weight average molecular weight calculation is averagedependant on the summation of all slices of the concentration andmolecular weight. The weight average molar weight can be measured, e.g.,using the Wyatt Astra software or any appropriate software. The weightaverage molecular weights described herein are determined by high liquidchromatography with two columns in series, for example a TSK G3000 SWXLand a G2000 SWXL, coupled with a multi angle light scattering (MALS)detector and a refractometric detector in series. The eluent used is a0.2 sodium sulfate, pH 5.0, and a flow rate of 0.5 mL/min.

Non-Reducing End Structure

In addition to chain length about 5 to 15 mole %, 7 to 14 mole %, or 9to 12 mole % of the chains in a preparation can haveΔUH_(NAc,6S)GH_(NS,3S,6S) at, or within about two, four or sixmonosaccharides from the non-reducing end of the chain. Methods that canbe used to quantify this structure include, e.g., capillaryelectrophoresis (CE) and high performance liquid chromatography (HPLC),e.g., reverse phase high performance liquid chromatography (RPHPLC). Toquantify the mole % of ΔUH_(NAc,6S)GH_(NS,3S,6S) in a LMWH preparation,a response factor (RF) for ΔUH_(NAc,6S)GH_(NS,3S,6S) can be determined.The determination can also include determining the RF for all speciesobtained, e.g., using CE or HPLC, e.g., a CE method described herein. Toobtain the RF for a species or all species obtained by CE, e.g., a CEmethod described herein, known concentrations of a standard for thespecie or one or more of the species can be injected on the CE and usedto determine a RF for each. The RF can then be used to determine themole %. As described herein, the sample has an actual level of astructure, which can be expressed, e.g., as 5 to 15 when described inunits of mole %. That actual level can also be expressed in other units,e.g., weight %. That actual level is the same regardless of the units inwhich it is expressed. The specification of mole % in the method ismerely to indicate the actual prevalence of the structure. The level ofstructure can be measured in terms of other units and the referencevalue can be expressed in terms of other units, as long as the referencevalue as expressed in terms of alternative units corresponds to the sameamount of structure as the reference value expressed in mole %, 5 to 15mole % in this example. Thus, a method which requires showing thestructure is present at 5 to 15 mole % can be performed by showing thatthe structure is present in a range expressed in an alternative unit ofmeasure, e.g., weight %, chain number, or % AUC, wherein the range, asdescribed in the alternative unit of measure, corresponds to the sameamount of the structure which would give the mole % referred to, in thisexample 5 to 15 mole %.

A LMWH preparation described herein can have a mixture of ΔU andiduronic acid (I)/glucuronic acid (G) at the non-reducing end of thechains in the preparation. The nomenclature “-U” refers to anunsaturated uronic acid (iduronic acid (I), glucuronic acid (G) orgalacturonic acid) that has a double bond introduced at the 4-5 positionas a result, e.g., of the lyase action of a heparinase, a HSGAG lyase,or other enzyme having similar substrate specificity. Preferably, about15% to 35%, 20 to 30% (e.g., 15%, 20%, 25%, 30%, 35%) of the totalnumber of chains in the preparation have a ΔU at the non-reducing end ofthe chain. The quantity of ΔU and/or I/G at the non-reducing end ofchains within the sample can be determined using, e.g., 2D-NMR. In suchmethods, the total number of chains having an acetylated hexosamine(H_(NAc)) at the reducing end and/or the number of open ringconfirmations at the reducing end can be used to determine the totalnumber of chains within the preparation. The total percentage of chainshaving a ΔU and/or I/G at the non-reducing end can be compared to thetotal number of chains in the preparation. Preferably, in the LMWHpreparations described herein, less than 90%, 95%, 98%, 99% or none ofthe chains in the preparation have a sulfated ΔU at the non-reducingend.

Reducing End Structures

In some instances, a LMWH preparation provided herein has substantiallyno modified reducing end structures. In preferred embodiments at least85%, 90%, 95%, 98%, 99% or all of the chains in the LMWH preparationhave a non-modified reducing end structure.

A “modified reducing end structure” refers to a structure that arises atthe reducing end of chains in the preparation due to the process ofisolating or preparing the preparation from natural sources. Forexample, many commercially available LMWH preparations are derived fromunfractionated heparin primarily through chemical or enzymaticdepolymerization of the polysaccharide chains. A process used to make aLMWH can cause one or more unique structural modifications to thereducing end of polysaccharide chains of starting material from anatural source. For example, nitrous acid depolymerization of heparinresults in the formation of a 2,5-anhydromannose at the reducing end,which can be reduced to form an alcohol, and depolymerization throughesterification of the carboxylate functional group on the uronic acidfollowed by υ-elimination results in the formation of a 1,6-anhydrostructures at the reducing end of some chains. Thus, 2,5-anhydromannoseand 1,6 anhydro structures are examples of modified reduce endstructures that can be found on some chains of LMWHs. The chains in aLMWH preparation provided herein can include, e.g., at least about 60%,70%, 80%, 85%, 90%, 95%, 98%, 99% or all of the chains having anacetylated hexosamine at the reducing end.

Anti-Xa Activity

Anti-Xa activity of a LMWH preparation plays a role in biologicalactivity of LMWH preparations. Preferably, a LMWH preparation providedherein has an anti-Xa activity of about 100 to 400 IU/mg, e.g., about120 to 380 IU/mg, e.g., about 150 to 350 IU/mg, e.g., about 170 to 330IU/mg, e.g., about 180 to 300 IU/mg, e.g., about 150 to 200 IU/mg, 200to 300 IU/mg. Anti-Xa activity of a LMWH preparation is calculated inInternational Units of anti-factor Xa activity per milligram using thestatistical methods for parallel line assays. The anti-factor Xaactivity of LMWH preparations described herein is measured using thefollowing principle:

M118+ATIII→[M118·ATIII]

FXa

M118·ATIII→[M118·ATIII·FXa]+FXa (Excess)

FXa (Excess)+Substrate→Peptide+pNA (measured spectrophotometrically)

The anti-factor Xa activity is determined by the sample potentiatingeffect on antithrombin (ATIII) in the inhibition of activated Factor Xa(FXa). Factor Xa excess can be indirectly spectrophotometricallymeasured. Anti-factor Xa activity can be measured, e.g., on aDiagnostica Stago analyzer with the Stachrom® Heparin Test kit, on anACL Futura3 Coagulation system with the Coatest® Heparin Kit fromChromogenix, or on any equivalent system. Analyzer response can becalibrated using the NIBSC International Standard for Low MolecularWeight Heparin.

Anti-Xa/IIa Ratio

In some aspects, LMWH preparations provided herein have an anti-Xaactivity to anti-IIa activity ratio of 3:1 or less, e.g., 2.1, 1.6:1,1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1. Methods of determininganti-factor Xa activity and the anti-factor IIa activity have beendescribed above. The ratio of anti-factor Xa activity to anti-factor IIaactivity is calculated by dividing anti-factor Xa activity (dry basis)by the anti-factor IIa activity (dry basis).

Both anti-Xa activity and anti-IIa activity of heparin and LMWHpreparations involve binding of antithrombin III (ATIII) to a specificsequence, represented by the structure ΔUH_(NAc,6S)GH_(NS,3S,6S), withinchains present in the preparation. Binding of ATIII to this sequencemediates anti-Xa activity. In addition, thrombin (factor IIa) bindsheparins at a site proximate to the ATIII binding site. Unlike anti-Xaactivity that requires only the ATIII binding site, anti-IIa activityrequires the presence of an ATIII binding site as well as a chain ofsufficient length distal to the ATIII binding site. The anti-IIaactivity of LMWH preparations provided herein can be attributed, atleast in part, to the presence of ΔUH_(NAc,6S)GH_(NS,3S,6S) at or nearthe non-reducing end of chains within the LMWH preparations as well asthe length of many of the chains present in the preparation. Thiscombination may result in chains within the preparation that contributeto both anti-Xa activity and anti-IIa activity. When both anti-Xaactivity and anti-IIa activity are provided by the same chain or chains,the clearance of that chain or chains can result in both a decrease inanti-Xa activity and anti-IIa activity. As such, the anti-Xa activityand anti-IIa activity can remain relatively constant over the course ofadministration. Therefore, in some aspects, the LMWH preparationsprovided herein have an anti-Xa activity to anti-IIa activity remainsrelatively constant over the course of an administration of LMWH, e.g.,the anti-Xa activity to anti-IIa activity ratio varies about +1.5, ±1,±0.5, or ±0.2, over a period of about 30, 60, 120, 180, 240, 300minutes. For example, if an initial ratio of anti-Xa activity t anti-IIaactivity is 2, then the ratio measured at a second time (e.g., 30, 60,120, 180, 240, 300 minutes) after the initial administration willpreferably be less than 3, and preferably at or around 2.

Neutralization

LMWH preparations provided herein can be neutralized by protaminesulfate. For example, anti-IIa activity and/or anti-Xa activity can beneutralized by at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100%by administration of protamine. Protamine sulfate is commerciallyavailable, e.g., from Eli Lilly and Company. Neutralization of anti-Xaactivity and anti-IIa activity can be measured, e.g., by standardcoagulation assays such as ACT and aPTT, both of which are describedfurther herein. Protamine sulfate can be administered intravenously,e.g., at a dose of about 1, 2, 3 mg per 100 anti-Xa IU of the LMWHpreparation in plasma. Preferably, protamine neutralization of anti-Xaactivity and/or anti-IIa activity occurs within 5, 10, 15, 20, 25, orminutes after administration of the protamine sulfate.

Polydispersity

The polydispersity of LMWH preparations provided herein is about 1.6 orless, e.g., about 1.6 or 1.5 to 1.1, and numbers in between.

The term “polydisperse” or “polydispersity” refers to the weight averagemolecular weight of a composition (Mw) divided by the number averagemolecular weight (Mn). The number average molecular weight (Mn) iscalculated from the following equation: Mn=Σci/(Σci/mi). The variable ciis the concentration of the polysaccharide in slice i and Mi is themolecular weight of the polysaccharide in slice i. The summations aretaken over a chromatographic peak, which contains many slices of data. Aslice of data can be pictured as a vertical line on a plot ofchromatographic peak versus time. The elution peak can therefore bedivided into many slices. The number average molecular weight is acalculation dependent on the molecular weight and concentration at eachslice of data. Methods of determining weight average molecular weightare described above, and were used to determine polydispersity as well.

For any of the ranges described herein, e.g., for a given structure oractivity, the ranges can be those ranges disclosed as well as otherranges. For example, a range constructed from a lower endpoint of onerange, e.g., for a given building block or activity, can be combinedwith the upper endpoint of another range, e.g., for the given buildingblock or activity, to give a range.

An “isolated” or “purified” LMWH preparation is substantially free ofcellular material or other contaminating proteins from the cell ortissue source from which the LMWH is derived, or substantially free fromchemical precursors or other chemicals when chemically synthesized.“Substantially free” means that a preparation of LMWH is at least 50%pure (wt/wt). In a preferred embodiment, the preparation of LMWH hasless than about 30%, 20%, 10% and more preferably 5% (by dry weight), ofnon-heparin polysaccharides, proteins or chemical precursors or otherchemicals, e.g., from manufacture. These also referred to herein as“contaminants”. Examples of contaminants that can be present in a LMWHpreparation provided herein include, but are not limited to, calcium,sodium, heparinase enzyme (or other enzyme having similar substratespecificity), methanol, ethanol, chloride, sulfate, dermatan sulfate,and chondrotin sulfate.

Methods of Monitoring Activity of a LMWH Preparation

The activity of a LMWH preparation provided herein can be monitored bystandard anti-coagulation assays. Such assays include, e.g., ACT andaPTT, both of which are routinely practiced in hospitals andspecifically hospital operating rooms.

ACT is a test that is used to monitor the effectiveness of heparintherapy. The ACT can be done at the bedside, e.g., for patientsexperiencing pulmonary embolus, extracorporeal membrane oxygenation(ECMO) and hemodialysis. ACT is most often used before, during and aftersurgical intervention such as, e.g., cardiopulmonary bypass (CPB)surgery, PCI and stent placement. Reference value for the ACT can rangefrom between 70-180 seconds. However, for certain procedures such as CPBthe desired range can exceed 400-500 seconds. ACT utilizes negativelycharged particles for a determination of time to clot formation.Examples of various particles that can be used include celite, which hasa normal length of ACT being about 100 to 170 seconds; kaolin, which hasa normal length of ACT being about 90 to 150 seconds; and glassparticles, which have a normal length of ACT being about 190 to 300seconds. Suitable machines for measuring ACT include, e.g., Hemochronand Medtronic HemoTec.

In the aPTT (also referred to as “partial thromboplastin time” or “PTT”)test, a contact activator is used to stimulate the production of FactorXIIa by providing a surface for the function of high molecular weightkininogen, kallikrein and Factor XIIa. This contact activation isallowed to proceed for a specific period of time. Calcium is then addedto trigger further reactions and the time required for clot formation ismeasured. Phospholipids are required to form complexes, which activateFactor X and Prothrombin. APTT can be measured by the IL Test™ APTT-SP(liquid). Reference values for aPTT is about 25 to 35 seconds. Aprolonged aPTT indicates that clotting is taking longer than expected,e.g., due to a heparin or LMWH treatment.

Methods of Making LMWH Preparations

Various methods of making LMWH preparations, e.g., a LMWH preparationdescribed herein are also contemplated. For example, such methodsinclude a method of making a LMWH preparation having an average chainlength of about 8 to 16 or 9 to 16 disaccharides. The method includesproviding a precursor LMWH preparation having a chain length of lessthan 8 to 16 or 9 to 16 disaccharides, and processing the precursor LMWHpreparation to obtain a LMWH preparation having an average chain lengthof about 8 to 16 or 9 to 16 disaccharides. Preferably, the precursor hasan average chain length of about 8 to 14, e.g., 8 to 12, disaccharides.For example, the precursor LMWH preparation can have the followingstructure:

wherein X is a monovalent or divalent cation (e.g., Na or Ca),

R is H or SO₃X; R1 is SO₃X or COCH₃;

n=2-45, e.g., 2-35;and the composition preferably has an average value for n of 7 to 13,e.g., 7 to 11, or 8 to 12.

A precursor LMWH preparation used in this method can be obtained by amethod that includes salt precipitation followed by (and) enzymaticdigestion. A salt of a monovalent or divalent cation can be used in themethod of obtaining the precursor LMWH preparation. Examples ofmonovalent and divalent cations that can be used include, e.g., sodium,potassium, rubidium, cesium, barium, calcium, magnesium, strontium, andcombinations thereof. The salt can be, e.g., an acetate of a monovalentor divalent cation. Enzymatic digestion to obtain the LMWH precursor caninclude the use of one or more enzymes that cleaves at one or moreglycosidic linkages of unsulfated uronic acids. Exemplary enzymesinclude heparinase III, mutants of heparinase III and HSGAG lyase IIIfrom Bacteroides thetaiotaomicron. Heparinase III is described, forexample, in U.S. Pat. Nos. 5,681,733 and 5,919,693. Mutants ofheparinase III are described in U.S. Pat. No. 5,896,789. Preferredheparinase III mutants are those mutants having one or more histidine atHis36, His105, His110, His139, His152, His225, His 234, His424, His469and His539 substituted with an alanine.

The precursor LMWH preparation can be processed by size dependentseparation such as, e.g., size exclusion chromatography, ion exchangechromatography and filtration. Further processing steps can be usedprior to or after the size dependant separation, e.g., to obtain drugproduct.

The term “drug product” refers to a LMWH preparation having the purityrequired for and being formulated for pharmaceutical use.

The term “drug substance” refers to a LMWH preparation having thepolysaccharide constituents for pharmaceutical use but is notnecessarily in its final formulation and/or comprises one or morenon-product contaminant (e.g., one or more inorganic product such assulfate, chloride, protein contaminant, process by-product such asheparinase, calcium, sodium).

Other methods of making a LMWH preparation as provided herein includesproviding a “fast moving fraction” from a glycosaminoglycan (GAG)containing sample, e.g., UFH. The fast moving fraction can be made asfollows:

(1) subjecting a GAG containing sample, e.g., UFH, to a first aprecipitation, e.g., with a polar organic solvent (e.g., an alcohol,e.g., ethanol), a polar non-organic solvent (e.g., water), and a salt(preferably, a sodium salt, e.g., sodium acetate), to yield a firstsupernatant;

(2) subjecting the first supernatant to a second precipitation, e.g.,with a polar organic solvent (e.g., an alcohol, e.g., ethanol), and apolar non-organic solvent (e.g., water), to yield a precipitate (thisprecipitate contains the fast moving fraction);

(3) and preferably solubilizing the precipitate.

Fractions of (GAG) containing sample, e.g., UFH made by other methods,but which produce a substantially equivalent fraction, e.g., one havingan average chain length of 9-16 disaccharides can also be used as a fastmoving fraction.

In some embodiments, the fast moving fraction has the followingstructure:

wherein,

X is Na or Ca;

R is H or SO₃Na;

R1 is SO₃Na or COCH₃;

n=2-50, e.g., 2-40;

and the composition preferably has an average value for n of 9 to 16 or8 to 15.

This composition can occur as an intermediate in the production of aLMWH, e.g., as the product of precipitations to provide a fast movingfraction (as discussed herein).

The fast moving fraction can be processed further to provide a LMWH ofthe invention. Processing of the fast moving fraction can includedigesting the fast moving fraction with a chemical or enzyme thatcleaves one or more glycosidic linkages of unsulfated uronic acid, e.g.,one or more glycosidic linkages of unsulfated uronic acid adjacent to anN-acetyl glucosamine residue, e.g., to give rise to a preparation withthe qualities and characteristics described herein. Enzymes can beevaluated for substrate specificity by the following steps: 1)functional screening of enzyme activity against two HSGAG substrateshaving different sulfation densities, e.g., heparin and heparan sulfate,whereby enzymes having a preference for heparan sulfate over heparin areselected; 2) fragment mapping of cleaved substrates from step 1 toassess substrate specificity; 3) cleavage of a LMWH such as M118-REHstep 1 intermediate or dalteparin using the enzyme, followed by; 4)assessment of anti-Xa activity and anti-IIa activity of the cleavedsubstrate using an in vitro assay; and 5) assessment of molecular weightdistribution (or average chain length) of cleaved substrate using gelpermeation chromatography (GPC) and/or size exclusion chromatographyinterfaced with multi-angle light scattering (SEC-MALS).

Step 1 assesses an enzyme's ability to act as an HSGAG lyase identifiedby the ability to generate an unsaturated C4-C5 bond at non-reducingends of cleavage products as well as the enzymes preference forundersulfated substrates such as heparan sulfate. Enzyme activity can befollowed spectrophotometrically by monitoring UV absorbance at 232 nm.An absorbance at this wavelength indicates formation of unsaturateduronic acids at the non-reducing ends of the cleavage product. Enzymeactivity is monitored both kinetically (initial rate of productformation) and in terms of total product formation following exhaustivedigestion (about 12 to 15 hours). Preferred enzymes have about a twofold preference for heparan sulfate over heparin and greater than a twofold (e.g., a 3 to 5 fold) difference in total activity.

The second step assesses the cleavage specificity of the enzyme. Enzymessuitable for making the LMWH compositions described hereinpreferentially cleave undersulfated regions of heparin or heparansulfate. If UFH is the substrate used, this preference is demonstratedby an obvious underdigestion of substrate (as indicated by the presenceof longer oligosaccharides) with any disaccharides being produced havinga low sulfate density. In contrast when the substrate is heparansulfate, digestion results in a greater number of disaccharides whichindicates a higher cutting frequency.

The remaining steps 3-5 can be performed as described elsewhere herein.

Examples of enzymes include heparinase III, mutants of heparinase IIIand HSGAG lyase from Bacteroides thetaiotaomicron. In some embodiments,the fast moving fraction is processed, at least in part, with a mutatedheparinase III having an alanine at residue 225 of the amino acidsequence of heparinase III instead of a histidine. This enzyme is alsoreferred to herein as “MO11”.

The digested LMWH preparation can be the final product, e.g., the drugsubstance or drug product, or can be further processed to obtain thefinal product, e.g., drug substance or drug product. The concentratedLMWH preparation can be further processed, e.g., by one or more of sizedependant separation (e.g., by size exclusion chromatography, ionexchange chromatography and filtration), and filtration. Preferably, theconcentrated LMWH preparation is further processed by a size dependantseparation, and the LMWH preparation obtained from this step has anaverage chain length of about 9 to 16 disaccharides.

Methods of Evaluating or Processing LMWH Preparations

Capillary Electrophoresis

Enzymes

Analysis of a LMWH preparation such as an M118-REH preparation using CEincludes, e.g., digesting the preparation with one or more heparindegrading enzymes. The heparin degrading enzyme(s) can be, e.g., one ormore heparinase, heparin lyase, HSGAG lyase, a lyase described as a GAGlyase that can also degrade heparin, and/or any polypeptide described asa hydrolase, sulfatase/sulfohyrdolase, or glycosylhydrolase/glycosidase. For example, the LMWH preparation can be digestedwith one or more of: an unsaturated glucuronyl hydrolase (e.g., F.heparinum Δ4,5 glycuronidase, B. thetaiotaomicron Δ4,5 glycuronidase); aglucuronyl hydrolase (e.g., mammalian α-iduronidase, β-glucuronidase); asulfohydrolase (e.g., F. heparinum 2-O-sulfatase, 6-O-sulfatase,3-O-sulfatase, B. thetaiotaomicron 6-O-sulfatase, a mucin desulfatingenzyme, mammalian N-acetylglucosamine-6-sulfatase, mammalian iduronicacid-2-sulfatase); a N-sulfamidase (e.g., F. heparinum N-sulfamidase,mammalian heparan-N-sulfatase); an arylsulfatase; a hexosaminidase; aglycosyl hydrolase (e.g., endo-N-acetyl glucosaminidase); a heparinase(e.g., Flavobacterum heparinum heparinase I, Flavobacterum heparinumheparinase II, Flavobacterum heparinum heparinase III, Flavobacterumheparinum heparinase IV); an endoglucoronidase (e.g., mammalianheparanase); a heparin/heparan sulfate lyase (e.g., Bacteroidesthetaiotaomicron HSGAG lyase I, Bacteroides thetaiotaomicron HSGAG lyaseII, Bacteroides thetaiotaomicron HSGAG lyase III, Bacteroidesthetaiotaomicron GAG lyase IV); and functional fragments and variantsthereof. It can also include a polypeptide described as above (e.g., aheparinase or a heparin/heparin sulfate lyase) derived frommicroorganisms other than Flavobacterium heparinum (a.k.a. Pedobacterheparinus) or Bacteroides thetaiotaomicron. For example, Haloarculamarismortui, Agrobacterium tumefaciens, Streptococcus pneumoniae,Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcusagalactiae, Streptococcus intermedius, Streptococcus suis, Enterococcusfaecalis, Rhodopseudomonas palustris, Nitrobacter winogradskyi,Nitrobacter hamburgensis, Bradyrhizobium japonicum, Rhizobium meloliti,Mesorhizobium loti, Spinghobacterium sp., Brucella abortus biovar,Brucella melitensis, Solibacter usitatus, Acidobacterium capsulatum,Microbulbifer degradans, Pseudomonas aeruginosa, Burkholderiapseudomonascepacia, Geobacter metallireducens, Prevotella sp., Serratamarcescens, Cornybacterium sp., Anaeromyxobacter dehalogenans,Rhodopirellula Baltica, Pirellula marina, and/or Gemmata obscuriglobus.

Preferably, at least one enzyme used in the digestion is selectedbecause it cleaves at specific linkages within heparins. For example,the enzyme can be heparinase I and/or HSGAG lyase I. In one embodiment,the LMWH preparation is digested with Flavobacterium heparinumheparinase I. In other embodiments, the heparin preparation is digestedwith Bacteroides thetaiotaomicron HSGAG lyase I.

Other enzymes can be selected for use in the digestion which resolvestructures which could not be resolved solely with the use of heparinaseI, II, and III. Any of the enzymes described herein can be replaced withan enzyme with functionally equivalent activity.

In a preferred embodiment, the digestion is run to completion or atleast sufficiently to provide a digest having all of the products foundin Table 10A and preferably substantially free of undigested material.

Prior to digestion, the sample can be lyophilized. For example, thesample can be dried in a vacuum oven, e.g., at about 20° C., 25° C., 30°C., 35° C., 40° C., 43° C., 46° C., 49° C., 52° C., or 55° C., for about2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours. For example, thesample can be lyophilized and/or dried under one of the followingconditions: For example, the sample can be lyophilized and/or driedunder one of the following conditions: 40° C. for 12 hours; 46° C. for 8hours; 49° C. for 6 hours; 52° C. for 4 hours. A sample can be suspendedin water or a suitable buffer (e.g., 1 mM calcium acetate, 25 mM sodiumacetate, pH 7.0, and 5% glycine) at a concentration of about 1, 2, 5,10, 20, 50, 100, 200, or 500 mg/mL. One or more heparin degrading enzymecan be added to the sample. In some embodiments, heparinase I or HSGAGlyase I (or combinations of these enzymes), heparinase II or HSGAG lyaseII (or combinations of these enzymes), and heparinase III or HSGAG lyaseIII (or a combination of these enzymes) are added to the sample. Thesample is digested at a temperature of about 18° C., 25° C., 30° C., 37°C., or 45° C. for about 6, 12, 16, 18, 20 or 24 hours, e.g., at about25° C. for 24 hours; at 30° C. for about 18 hours; at about 37° C. for12 hours.

Following digestion, the enzyme or enzymes are removed from the samplemixture, e.g., using a Ni²⁺ column, a size-exclusion column, dialysis,ultrafiltration, or the like. The enzyme or enzyme can be inactivated byheating (e.g., at 65° C. for 20 minutes) following digestion. The samplecan be stored, e.g., at −85° C., −70° C., −20° C., 4° C., 18° C., or 25°C. for a period of time prior to analysis.

Species separated by the methods described herein can be detected bynumerous means, e.g., by ultraviolet absorbance (e.g., at a wavelengthof about 232 nm), evaporative light scattering, fluorescence, pulsedamperometric detection, and mass spectrometry. In some embodiments, twoor more means of detection can be utilized on the same sample, e.g., inseries or in parallel.

Additional enzyme digestions can be used to digest the sample. Forexample, a combination of heparinase I or HSGAG lyase I (or combinationsof these enzymes), heparinase II or HSGAG lyase II (or combinations ofthese enzymes), heparinase III or HSGAG lyase III (or a combination ofthese enzymes), and 2-0 sulfatase, Δ4,5 glycuronidase, and/or heparinaseI or HSGAG lyase I (or combinations of these enzymes), heparinase II orHSGAG lyase II (or combinations of these enzymes), heparinase III orHSGAG lyase III (or a combination of these enzymes), can be used fordigestion, and, e.g., detected by the methods described above.

The digestion products are analyzed using an Agilent 3D CapillaryElectrophoresis instrument. The capillary is an extended light path barefused-silica capillary 75 μm ID, effective length 72 cm. Tris (50 mM),10 μM dextran sulfate at pH 2.5 is used as CE buffer. Samples areinjected at a pressure of 30 mbar for 20 seconds. Separation isperformed at negative polarity and the analyte is monitored at 232 nmwith 310 nm as the reference wavelength. New capillaries are pre-treatedwith a sequence of water, 1N sodium hydroxide, water, and separationbuffer. For each sample analysis, the capillary is preconditioned withbuffer for 5 minutes.

Additional information useful for the methods described herein can befound in, e.g., Linhardt et al. (1988) Biochem. J., 254:781-787; Chuanget al. (2001) J. Chromatogr. A, 932:65-74; and Yates et al. (2004) J.Med. Chem., 47:277-280, and Rhomberg et al. (1988) Proc Natl Acad SciUSA. 95(8):4176-81.

Capillary Electrophoresis

CE, using e.g., an uncoated fused silica capillary, can be used toanalyze LMWH, e.g., LMWH preparations described herein. Under conditionsof low pH, separation is dictated by analyte electrophoretic mobilityalmost exclusively. Due to the fact that all LMWH related saccharideshave a net negative charge due to the carboxylate and sulfate moieties,separation is conducted under reverse polarity. In addition,supplementation of the low pH (pH2.5) buffer with dextran sulfateprevents non-specific absorption of anionic heparin-like material,enabling symmetrical peaks shapes and accurate quantification.

The species in LMWH preparation can be resolved with a series of fivedigests (discussed in detail elsewhere herein); each digest is subjectedto capillary electrophoresis after addition of an internal standardnaphthalene monosulfonate.

14 individual components (see, e.g., Table 10, herein) are resolved inthe CE.

Mass recovery in the compositional analysis methodology was evaluated asfollows. This analysis occurred at two levels: (1) mass recovery afterenzymatic digestion and (2) mass recovery after separation withcapillary electrophoresis.

NMR

Two dimensional nuclear magnetic resonance spectroscopy (2D NMR) can beused as a means of partially resolving and identifying signals withminimum signal overlap. Integration of the 2D NMR signals followed bysimple calculations can facilitate a quantitative monosaccharidecompositional analysis of a polysaccharide mixture such as analysis of aLMWH preparation such as those provided herein.

Moreover, 2D NMR can provide information on linkage environments of adisaccharide constituent, for example an H-U disaccharide, providinganalysis of disaccharide linkages, including both qualitative andquantitative analysis. In some embodiments, 2D NMR analysis can provideinformation about the epimerization state of a H-U linkage, for example,providing information as to whether the epimerization state is aniduronic acid residue or an glucuronic acid residue (i.e., I or U).

In some embodiments, a 2D proton-carbon correlation spectroscopy (HSQC)experiment can provide quantitative compositional analysis on one ormore glycosaminoglycan. For example, in some embodiments 2D NMR analysiscan provide information about the nearest neighbor at the reducing endof a monosaccharide. This information can provide, for example, thesequence context in which a particular monosaccharide is present in apolysaccharide mixture, e.g., a LMWH such as a LMWH preparationdescribed herein.

In some embodiments, 2D NMR method allows to discriminate betweeninternal and reducing end residues. In particular, identification ofmeasurable amounts of reducing N-acetyl glucosamine is peculiar of thoseLMWH described herein.

In some embodiments, 2D NMR analysis can provide information about thenon-reducing end of LMWH chains, i.e. the amount of ΔΔAp2-OH generatedby the enzymatic digestion.

In some embodiments, 2D NMR can be used to evaluate a polysaccharidemixture for the presence of one or more impurities such as dermatansulfate. For example, the absence of a signal in the proton NMR at2.06-2.09 ppm can be used to confirm that dermatan sulfate is notpresent at levels greater than the level of detection of the instrument(e.g., at a level greater than about 1%).

In a preferred embodiment saccharide structure is evaluated using, 2DNMR, e.g., e.g., sample of a polysaccharide mixture exchanged with D2O,lyophilized over night, and redissolved in D2O. The sample is thenplaced in an NMR tube for analysis and run at 303 K with a Bruker Avance600 MHz spectrometer equipped with a 5-mm TXI probe. Gradient-enhancedHSQC spectra is recorded with carbon decoupling during acquisition. Thedata is then acquired with 16 scans for each of 256 increments in theindirect 13C dimension. The polarization transfer delay is set to 2.941ms for an optimal transfer with 1JCH scalar couplings of 155 Hz.

The data is generally then processed, e.g. the matrix size 1K×256 iszero filled to 2K×1K by application of a squared-cosine function priorto Fourier transformation. Cross peaks are integrated, for example,using MestreC 4.5 software and only positive peaks are used forintegration. Integrals are normalized to the H2/C2 peak of N-sulfatedglucosamine (3.26/60.5 ppm). Peaks are generally assigned usingpublished chemical shifts and experimental assignments via COSY andTOCSY experiments.

Percent composition is calculated using the anomeric cross peak volumes,for which all uronic acid residues have similar 1JCH couplings as do allglucosamine residues. For glucosamine anomeric peaks where overlappingprevents precise quantification, H2/C2 signals are integrated instead.The amount of every monosaccharide is expressed as percentage of thetotal glucosamine or uronic acid content. The ratio of 6-O-sulfationversus 6-O-desulfation is calculated from H6/C6 signal integration.

The percent composition data is provided in Table 11A.

Additional information useful for the methods found herein can be foundin, e.g. Guerrini et al. (2005) Anal. Biochem., 337: 35-47.

Pharmaceutical Compositions

Compositions, e.g., pharmaceutically acceptable compositions, whichinclude a LMWH preparation described herein, formulated together with apharmaceutically acceptable carrier, are provided.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, isotonic and absorption delaying agents,and the like that are physiologically compatible. The carrier can besuitable for intravenous, intramuscular, subcutaneous, parenteral,rectal, spinal or epidermal administration (e.g., by injection orinfusion).

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, liposomes and suppositories. The preferred form dependson the intended mode of administration and therapeutic application.Typical preferred compositions are in the form of injectable orinfusible solutions. The preferred mode of administration is parenteral(e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In apreferred embodiment, the LMWH preparation is administered byintravenous infusion or injection. In another preferred embodiment, theLMWH preparation is administered by intramuscular or subcutaneousinjection.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intravitreous, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion.

Therapeutic compositions typically should be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, dispersion, liposome, or otherordered structure suitable to high concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (I.e.,LMWH in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying that yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof. The proper fluidity of a solution canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, various polymers,monostearate salts and gelatin.

The LMWH preparations can be administered by a variety of methods knownin the art, although for many therapeutic applications, the preferredroute/mode of administration is intravenous injection or infusion. Aswill be appreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results. In certainembodiments, the active compound may be prepared with a carrier thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multi-dose containers, e.g., with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents.

In certain embodiments, a LMWH preparation provided herein can be orallyadministered, for example, with an inert diluent or an assimilableedible carrier. The compound (and other ingredients, if desired) mayalso be enclosed in a hard or soft shell gelatin capsule, compressedinto tablets, or incorporated directly into the subject's diet. For oraltherapeutic administration, the compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.To administer a compound of the invention by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses. Pharmaceuticalcompositions which can be used orally include push-fit capsules made ofgelatin, as well as soft, sealed capsules made of gelatin and aplasticizer, such as glycerol or sorbitol. The push-fit capsules cancontain the active ingredients in admixture with filler such as lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. Microspheres formulated for oraladministration may also be used. Such microspheres have been welldefined in the art. All formulations for oral administration should bein dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the LMWH preparation may beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch. In addition, dry powder formations for inhalationtherapy are within the scope of the invention. Such dry powderformulations may be prepared as disclosed in WO 02/32406.

The composition may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the compositions described previously, the compounds mayalso be formulated as a depot preparation. Such long-acting formulationsmay be formulated with suitable polymeric or hydrophobic materials (forexample, as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude, but are not limited to, calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Examples of compositions which can be used for non-parental delivery(e.g., non-invasive delivery) include: metered amounts of a compositionto be administered from an inhaler for pulmonary delivery; tabletshaving a prescribed dosage unit for oral administration; transdermalpatches to deliver a dosage unit across the skin; and suppositories todeliver a desired dosage unit rectally or vaginally. The compositionscan be included in a container, pack, or dispenser together withinstructions for administration.

The LMWH preparation can also be administered with short or long termimplantation devices. The preparation can be implanted subcutaneously,can be implanted into tissues or organs (e.g., the coronary artery,carotid artery, renal artery and other peripheral arteries, veins,kidney, heart cornea, vitreous, cerebrum, etc.), or can be implanted inphysiological spaces around tissues and organs (e.g., kidney capsule,pericardium, thoracic or peritoneal space).

The LMWH preparation can also be used to coat various medical devices.For example, the LMWH preparation can be used to coat a stent orextracorporeal circuit. Such formulations of the LMWH preparations mayinclude using, e.g., controlled release beads, gel or microspheres aswell as various polymers such as PLGA, cellulose, alginate or otherpolysaccharides.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated. It is to be further understood thatfor any particular subject, specific dosage regimens should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of a LMWH preparation. A “therapeutically effective amount”refers to an amount effective, at dosages and for periods of timenecessary, to achieve a desired therapeutic result. A therapeuticallyeffective amount of the LMWH preparation may vary according to factorssuch as the disease state, age, sex, and weight of the individual, andthe LMWH preparation to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the LMWH preparation is outweighed by thetherapeutically beneficial effects. A “therapeutically effective dosage”preferably inhibits a measurable parameter, e.g., coagulation orthrombosis, e.g., as measured by ACT and aPTT, by at least about 20%,more preferably by at least about 40%, even more preferably by at leastabout 60%, and still more preferably by at least about 80% relative tountreated subjects. The ability of a compound to inhibit a measurableparameter, e.g., coagulation or thrombosis, can be evaluated in ananimal model system predictive of efficacy in humans. Alternatively,this property of a composition can be evaluated by examining the abilityof the compound in an in vitro assay. Exemplary doses for intravenousadministration of the LMWH preparation are about IU/kg to 200 IU/kg,e.g., 1 IU/kg; 2 IU/kg; 3 IU/kg, 4 IU/kg, 5 IU/kg, 6 IU/kg, 7 IU/kg, 8IU/kg, 9 IU/kg, 10 IU/kg, 11 IU/kg, 12 IU/kg, 13 IU/kg, 14 IU/kg, 15IU/kg, 16 IU/kg, 17 IU/kg, 18 IU/kg, 19 IU/kg, 20 IU/kg, 21 IU/kg, 22IU/kg, 25 IU/mg, 30 IU/kg, 40 IU/kg, 50 IU/kg, 70 IU/kg, 100 IU/kg, 125IU/kg, 150 IU/kg, 175 IU/kg, 200 IU/kg. Other exemplary doses forintravenous administration of the LMWH preparation are about 0.03 mg/kgto 0.45 mg/kg, e.g., 0.03 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2mg/kg, 0.22 mg/kg, 0.25 mg/kg, 0.27 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.37mg/kg, 0.4 mg/kg, 0.44 mg/kg, preferably about 0.1 mg/kg, 0.15 mg/kg,0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg, 0.44 mg/kg,0.47 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.60 mg/kg, 0.7 mg/kg, preferablyabout 0.30 to 0.50 mg/kg, e.g., 0.30 mg/kg, 0.35 mg/kg, 0.40 mg/kg, 0.42mg/kg, 0.44 mg/kg, 0.47 mg/kg or 0.50 mg/kg.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Also within the scope of the invention are kits comprising a LMWHpreparation provided herein. The kit can include one or more otherelements including: instructions for use; other reagents, e.g., atherapeutic agent or protamine sulfate; devices or other materials forpreparing the LMWH preparation for administration; pharmaceuticallyacceptable carriers; and devices or other materials for administrationto a subject. Instructions for use can include instructions formonitoring anti-Xa activity and/or anti-IIa activity using coagulationassays such as ACT and aPTT. The instructions can include instructionsfor therapeutic application including suggested dosages and/or modes ofadministration, e.g., in a patient having a disorder, e.g., a disorderdescribed herein. Other instructions can include instructions onreversing anti-Xa activity and/or anti-IIa activity using protaminesulfate. The kit can further contain at least one additional reagent,such as a diagnostic or therapeutic agent, e.g., a diagnostic ortherapeutic agent as described herein, formulated as appropriate, in oneor more separate pharmaceutical preparations.

Prophylactic and Therapeutic Uses

The LMWH preparations can be used to treat a subject. As used herein,the term “treat” or “treatment” is defined as the application oradministration of a LMWH preparation to a subject, e.g., a patient, orapplication or administration to an isolated tissue or cell from asubject, e.g., a patient, which is returned to the patient. The subjectcan be a patient having a disorder (e.g., a disorder as describedherein), a symptom of a disorder or a predisposition toward a disorder.The treatment can be to cure, heal, alleviate, relieve, alter, remedy,ameliorate, palliate, improve or affect the disorder, the symptoms ofthe disorder or the predisposition toward the disorder. As used herein,a subject is a vertebrate such as a human, non-human primate, cow,horse, pig, sheep, goat, dog, cat, or rodent. The subject can be, e.g.,an experimental animal, a veterinary animal, or a human subject. Atreatment can be therapeutic, e.g., a treatment which cures, heals,alleviates, relieves, alters, remedies, ameliorates, palliates, improvesor affects the disorder or a symptom of the disorder, e.g., lessens,mitigates or ameliorates an existing unwanted condition or symptomthereof, or prophylactic, e.g., a treatment which delays, e.g.,prevents, the onset of an unwanted condition or symptom thereof.

Heparins and LMWHs have many therapeutic utilities. The LMWHpreparations provided herein can be used for the treatment of any typeof condition in which heparin or LMWH therapy is useful. Thus, thepreparations and methods are useful in a variety of in vitro, in vivoand ex vivo methods. For instance, it is known that heparins and LMWHsare useful for preventing and treating dementia, such as Alzheimer'sdisease, disorders associated with coagulation (e.g., DVT and PE),fibrotic disorders (e.g., major organ fibrosis, fibroproliferativedisorders and scarring associated with trauma), thrombotic disorders(e.g., ACS, stable or unstable angina, MI (e.g., STEMI and NSTEMI)) orcardiovascular disease (atherosclerosis), vascular conditions orarterial fibrillation, allergy or respiratory disorders (e.g., asthma,emphysema, adult respiratory distress syndrome (ARDS), cystic fibrosis,and lung reperfusion injury), circulatory shock and related disorders,angiogenic disorders, cancer and metastatic disorders, sepsis, stenosisand restenosis, and osteoporosis. The LMWH preparations provided hereincan also be used on subjects having a fracture (e.g., a hip fracture) orto a subject prior to, during or after a surgical intervention (e.g.,organ transplant, orthopedic surgery, hip replacement, knee replacement,PCI, stent placement, angioplasty and CABG). Each of these disorders iswell-known in the art and is described, for instance, in Harrison'sPrinciples of Internal Medicine (McGraw Hill, Inc., New York), which isincorporated by reference. The use of HLGAG compositions in varioustherapeutic methods is described and summarized in Huang, J. andShimamura, A., Coagulation Disorders, 12, 1251-1281 (1998).

Thus, the LMWH preparations are useful for treating or preventingdisorders associated with coagulation. When an imbalance in thecoagulation pathway shifts towards excessive coagulation, the result isthe development of thrombotic tendencies, which are often manifested asheart attacks, strokes, DVT, ACS, stable and unstable angina, andmyocardial infarcts. A “disease associated with coagulation” as usedherein refers to a condition characterized by local inflammation whichcan result from an interruption or reduction in the blood supply to atissue which may occur, for instance, as a result of blockage of a bloodvessel responsible for supplying blood to the tissue such as is seen formyocardial or cerebral infarction or peripheral vascular disease, or asa result of emboli formation associated with conditions such as arterialfibrillation, DVT or PE. Persons undergoing surgery, anesthesia andextended periods of bed rest or other inactivity are often susceptibleto a condition known as deep venous thrombosis, or DVT, which is aclotting of venous blood in the lower extremities and/or pelvis. Thisclotting occurs due to the absence of muscular activity in the lowerextremities required to pump the venous blood (stasis), local vascularinjury or a hypercoaguble state. The condition can be life-threateningif a blood clot migrates to the lung, resulting in a “pulmonary embolus”or otherwise interferes with cardiovascular circulation. One method oftreatment involves administration of an anti-coagulant.

The methods are useful for treating thrombotic disorders andcardiovascular disease. Cardiovascular disease includes, but are notlimited to, atherosclerosis and arterial fibrillation. Atrialfibrillation is a common form of arrhythmia generally arising as aresult of emotional stress or following surgery, exercise, or acutealcoholic intoxication. Arterial fibrillation is characterized bydisorganized arterial activity without discrete P waves on the surfaceECG. This disorganized activity can lead to improper blood flow in theatrium and thrombus formation. These thrombi can embolize, resulting incerebral ischemia and other disorders.

Thrombotic disorders include, but are not limited to, ACS, e.g., MI,stable and unstable angina. Myocardial infarction is a disease statewhich sometimes occurs with an abrupt decrease in coronary blood flowthat follows a thrombotic occlusion of a coronary artery previouslynarrowed by atherosclerosis. Such injury may be produced or facilitatedby factors such as cigarette smoking, hypertension, and lipidaccumulation. Angina is due to transient myocardial ischemia. Thisdisorder is usually associated with a heaviness, pressure, squeezing,smothering, or choking feeling below the sternum. Episodes are usuallycaused by exertion or emotion, but can occur at rest. STEMI, alsoreferred to as “Q wave myocardial infarction”, refers to MI with anabnormal echocardiogram. NSTEMI, or “non-Q wave myocardial infarction”,is not associated an echocardiogram abnormality. Stable angina occurs atpredictable times with a specific amount of exertion or activity.Unstable angina may occur as a change in the usual pattern of stableangina. It my include chest pain that occurs at rest or with less andless exertion, that may be more severe and last longer, or that is lessresponsive to nitroglycerin. Unstable angina means that blood flow hasgotten worse potentially by an increased narrowing or small blood clotsthat form in the coronary arteries. Unstable angina is a warning signthat myocardial infarction may soon occur.

The LMWH preparation can be used for the treatment of thrombotic andcardiovascular disorders alone or in combination with other therapeuticagents for reducing the risk of a cardiovascular disease or for treatingthe cardiovascular disease. For example, the combination therapy caninclude a LMWH preparation coformulated with, and/or coadministeredwith, one or more additional therapeutic agents, e.g., one or moretherapeutic agent described herein. Administered “in combination”, asused herein, means that two (or more) different treatments are deliveredto the subject during the course of the subject's affliction with thedisorder, e.g., the two or more treatments are delivered after thesubject has been diagnosed with the disorder or identified as at riskfor the disorder and before the disorder has been prevented, cured oreliminated. In some embodiments, the delivery of one treatment is stilloccurring when the delivery of the second begins, so that there isoverlap. This is sometimes referred to herein as “simultaneous” or“concurrent delivery.” In other embodiments, the delivery of onetreatment ends before the delivery of the other treatment begins. Thedelivery can be such that an effect of the first treatment delivered isstill detectable when the second is delivered. Other therapeutic agentsinclude, but are not limited to, anti-inflammatory agents,anti-thrombotic agents, anti-platelet agents, fibrinolytic agents,thrombolytics, lipid reducing agents, direct thrombin inhibitors,anti-Xa inhibitors, anti-IIa inhibitors, glycoprotein IIb/IIIa receptorinhibitors and direct thrombin inhibitors. Examples of agents that canbe administered in combination with the LMWH preparations providedherein include bivalirudin, hirudin, hirugen, Angiomax, agatroban,PPACK, thrombin aptamers, aspirin, GPIIb/IIIa inhibitors (e.g.,Integrelin), P2Y12 inhibitors, thienopyridine, ticlopidine, andclopidogrel.

The monitorability by standard anticoagulation assays such as ACT andaPTT as well as the reversibility of the LMWH preparations providedherein provided improved flexibility in treating patients such as thosepatients admitted to the hospital and undergoing evaluation for possiblecardiovascular surgery. Such benefits are highlighted by the followingscenario. A patient goes the hospital complaining of symptoms that canbe associated with various thrombotic disorders such as ACS includingstable angina, unstable angina and MI. The monitorability andreversibility of the LMWH preparations provided herein allow use of suchpreparations while the patient is being evaluated for potentialcardiovascular surgery. If it is determined that the patient willreceive surgical intervention such as PCI or stent placement, themonitorability of the LMWH preparations, the anti-Xa activity andanti-IIa activity of the LMWH preparation can be monitored during theprocedure, and, if necessary, one or more additional doses of the LMWHpreparation can be given during or after the procedure to maintain theseactivities. If it is determined that a patient will receive a surgicalintervention such as CABG, the anti-Xa activity and anti-IIa activity ofthe LMWH preparation can be neutralized with protamine sulfate prior tosurgical intervention. In addition, anti-Xa activity and anti-IIaactivity can be monitored in the patient to ensure the activity issufficiently decreased prior to the surgery.

The LMWH preparations provided herein are also useful for treatingvascular conditions. Vascular conditions include, but are not limitedto, disorders such as DVT, peripheral vascular disease, cerebralischemia, including stroke, and PE. A cerebral ischemic attack orcerebral ischemia is a form of ischemic condition in which the bloodsupply to the brain is blocked. This interruption or reduction in theblood supply to the brain may result from a variety of causes, includingan intrinsic blockage or occlusion of the blood vessel itself, aremotely originated source of occlusion, decreased perfusion pressure orincreased blood viscosity resulting in inadequate cerebral blood flow,or a ruptured blood vessel in the subarachnoid space or intracerebraltissue. The methods are useful for treating cerebral ischemia. Cerebralischemia may result in either transient or permanent deficits and theseriousness of the neurological damage in a patient who has experiencedcerebral ischemia depends on the intensity and duration of the ischemicevent. A transient ischemic attack is one in which the blood flow to thebrain is interrupted only briefly and causes temporary neurologicaldeficits, which often are clear in less than 24 hours. Symptoms of TIAinclude numbness or weakness of face or limbs, loss of the ability tospeak clearly and/or to understand the speech of others, a loss ofvision or dimness of vision, and a feeling of dizziness. Permanentcerebral ischemic attacks, also called stroke, are caused by a longerinterruption or reduction in blood flow to the brain resulting fromeither a thrombus or embolism. A stroke causes a loss of neuronstypically resulting in a neurologic deficit that may improve but thatdoes not entirely resolve.

Thromboembolic stroke is due to the occlusion of an extracranial orintracranial blood vessel by a thrombus or embolus. Because it is oftendifficult to discern whether a stroke is caused by a thrombosis or anembolism, the term “thromboembolism” is used to cover strokes caused byeither of these mechanisms.

The methods are also directed to the treatment of acute thromboembolicstroke using a LMWH preparation provided herein. An acute stroke is amedical syndrome involving neurological injury resulting from anischemic event, which is an interruption or reduction in the bloodsupply to the brain.

An effective amount of a LMWH preparation alone or in combination withanother therapeutic for the treatment of stroke is that amountsufficient to reduce in vivo brain injury resulting from the stroke. Areduction of brain injury is any prevention of injury to the brain whichotherwise would have occurred in a subject experiencing a thromboembolicstroke absent the treatment described herein. Several physiologicalparameters may be used to assess reduction of brain injury, includingsmaller infarct size, improved regional cerebral blood flow, anddecreased intracranial pressure, for example, as compared topretreatment patient parameters, untreated stroke patients or strokepatients treated with thrombolytic agents alone.

The LMWH preparation may be used alone or in combination with atherapeutic agent for treating a disease associated with coagulation.Examples of therapeutics useful in the treatment of diseases associatedwith coagulation include anticoagulation agents, antiplatelet agents,and thrombolytic agents.

Anticoagulation agents prevent the coagulation of blood components andthus prevent clot formation. Anticoagulants include, but are not limitedto, warfarin, Coumadin, dicumarol, phenprocoumon, acenocoumarol, ethylbiscoumacetate, and indandione derivatives. “Direct thrombin inhibitors”include hirudin, hirugen, Angiomax, agatroban, PPACK, thrombin aptamers.Antiplatelet agents inhibit platelet aggregation and are often used toprevent thromboembolic stroke in patients who have experienced atransient ischemic attack or stroke. Thrombolytic agents lyse clotswhich cause the thromboembolic stroke. Thrombolytic agents have beenused in the treatment of acute venous thromboembolism and pulmonaryemboli and are well known in the art (e.g. see Hennekens et al, J AmColl Cardiol; v. 25 (7 supp), p. 18S-22S (1995); Holmes, et al, J AmColl Cardiol; v.25 (7 suppl), p. 10S-17S (1995)).

Pulmonary embolism as used herein refers to a disorder associated withthe entrapment of a blood clot in the lumen of a pulmonary artery,causing severe respiratory dysfunction. Pulmonary emboli often originatein the veins of the lower extremities where clots form in the deep legveins and then travel to lungs via the venous circulation. Thus,pulmonary embolism often arises as a complication of deep venousthrombosis in the lower extremity veins. Symptoms of pulmonary embolisminclude acute onset of shortness of breath, chest pain (worse withbreathing), and rapid heart rate and respiratory rate. Some individualsmay experience haemoptysis.

The preparations and methods are also useful for treating or preventingatherosclerosis. Heparin has been shown to be beneficial in preventionof atherosclerosis in various experimental models. Atherosclerosis isone form of arteriosclerosis that is believed to be the cause of mostcoronary artery disease, aortic aneurysm and atrial disease of the lowerextremities, as well as contributing to cerebrovascular disease.

The LMWH preparations are also useful before, during or after surgicaland dialysis procedures. Surgical patients, especially those over theage of 40 years have an increased risk of developing DVT. Thus, the useof the LMWH preparations provided herein for preventing the developmentof thrombosis associated with surgical procedures is contemplated. Inaddition to general surgical procedures such as percutaneousintervention (e.g., percutaneous coronary intervention (PCI)), PCTA,stents and other similar approaches, hip or knee replacement,cardiac-pulmonary by-pass surgery, coronary revascularization surgery,orthopedic surgery, and prosthesis replacement surgery, the methods arealso useful in subjects undergoing a tissue or organ transplantationprocedure or treatment for fractures such as hip fractures.

In addition, the LMWH preparations provided herein are useful fortreatment of respiratory diseases such as cystic fibrosis, asthma,allergy, emphysema, adult respiratory distress syndrome (ARDS), lungreperfusion injury, and ischemia-reperfusion injury of the lung.

Cystic fibrosis is a chronic progressive disease affecting therespiratory system. One serious consequence of cystic fibrosis isPseudomonas aeruginosa lung infection, which by itself accounts foralmost 90% of the morbidity and mortality in cystic fibrosis.Therapeutics for treating cystic fibrosis include antimicrobials fortreating the pathogenic infection.

Asthma is a disorder of the respiratory system characterized byinflammation, narrowing of the airways and increased reactivity of theairways to inhaled agents. Asthma is frequently, although notexclusively, associated with atopic or allergic symptoms. Asthma mayalso include exercise induced asthma, bronchoconstrictive response tobronchostimulants, delayed-type hypersensitivity, auto immuneencephalomyelitis and related disorders. Allergies are generally causedby IgE antibody generation against allergens. Emphysema is a distentionof the air spaces distal to the terminal bronchiole with destruction ofalveolar septa. Emphysema arises out of elastase induced lung injury.Adult respiratory distress syndrome is a term which encompasses manyacute defuse infiltrative lung lesions of diverse ideologies which areaccompanied by severe atrial hypoxemia. One of the most frequent causesof ARDS is sepsis.

Inflammatory diseases include but are not limited to autoimmune diseasesand atopic disorders. Other types of inflammatory diseases which aretreatable with the LMWH preparations provided herein are refractoryulcerative colitis, Crohn's disease, multiple sclerosis, autoimmunedisease, non-specific ulcerative colitis, sepsis and interstitialcystitis.

The LMWH preparations can be used to treat fibrotic disorders such asmajor organ fibrosis, fibroproliferative disorders and scarringassociated with trauma. Major organ fibrosis includes, but is notlimited to, interstitial lung disease (ILD), liver cirrhosis, kidneydisease (e.g., diabetes and untreated hypertensive disease), heartdisease and disorders of the eye (e.g., macular degeneration, retinal orvitreous retinopathy). Examples of Fibroproliferative disorders includesystemic and local scleroderma, keliods and hypertrophic scars,atherosclerosis, restenosis, fibrosarcoma and rheumatoid arthritis.Examples of scarring associated with trauma include scarring due tosurgery, chemotherapeutic-induced fibrosis, radiation-induced fibrosis,scarring associated with injury or burns.

In one embodiment, the LMWH preparations are used for inhibitingangiogenesis. Angiogenesis as used herein is the inappropriate formationof new blood vessels. “Angiogenesis” often occurs in tumors whenendothelial cells secrete a group of growth factors that are mitogenicfor endothelium causing the elongation and proliferation of endothelialcells which results in the generation of new blood vessels. Several ofthe angiogenic mitogens are heparin binding peptides which are relatedto endothelial cell growth factors. The inhibition of angiogenesis cancause tumor regression in animal models, suggesting a use as atherapeutic anticancer agent. Angiogenic disorders include, but are notlimited to, neovascular disorders of the eye, osteoporosis, psoriasis,arthritis, cancer and cardiovascular disorders.

The LMWH preparations, may also be used inhibit cancer cell growth andmetastasis. Thus the methods are useful for treating and/or preventingtumor cell proliferation or metastasis in a subject. The cancer may be amalignant or non-malignant cancer. Cancers or tumors include but are notlimited to biliary tract cancer; brain cancer; breast cancer; cervicalcancer; choriocarcinoma; colon cancer; endometrial cancer; esophagealcancer; gastric cancer; intraepithelial neoplasms; leukemias, lymphomas;liver cancer; lung cancer (e.g. small cell and non small cell);melanoma; neuroblastomas; oral cancer; ovarian cancer; pancreaticcancer; prostate cancer; rectal cancer; sarcomas; skin cancer;testicular cancer; thyroid cancer; and renal cancer, as well as othercarcinomas and sarcomas.

A subject in need of cancer treatment may be a subject who has a highprobability of developing cancer. These subjects include, for instance,subjects having a genetic abnormality, the presence of which has beendemonstrated to have a correlative relation to a higher likelihood ofdeveloping a cancer and subjects exposed to cancer-causing agents suchas tobacco, asbestos, or other chemical toxins, or a subject who haspreviously been treated for cancer and is in apparent remission.

When administered to a patient undergoing cancer treatment, the LMWHpreparation may be administered in cocktails containing otheranti-cancer agents. The LMWH preparation may also be administered incocktails containing agents that treat the side-effects of radiationtherapy, such as anti-emetics, radiation protectants, etc.

The treatments provided herein can further include administeringprotamine sulfate to neutralize the anti-Xa activity and/or anti-IIaactivity of the LMWH preparation, e.g., once anti-coagulation oranti-thrombotic activity is no longer necessary. Protamine sulfate canbe administered, e.g., by intravenous administration, at a dose of about1, 2, 3 mg of protamine sulfate per 100 IU of anti-Xa activity. The IUsof anti-Xa activity can be determined using, e.g., the coagulationassays described herein.

Other Embodiments

This invention is further illustrated by the following examples thatshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are incorporated herein by reference.

Examples Methods of Manufacturing M118-REH

Manufacturing Process

The depiction of the process used to produce M118-REH is shown inFIG. 1. Briefly, in Step 1 of the process, commercially availableUnfractionated Heparin, USP (UFH) was subjected to a step-wise series ofaqueous ethanol precipitations with calcium acetate in a 3:1 mass tomass ratio of calcium acetate to UFH to extract the portion of the UFHthat is of lower molecular weight (also referred as the portion that issubstantially the fast moving fraction). The resulting product of Step 1fractionation was designated Intermediate 1.

Step 2 involved the digestion of Intermediate 1 using a modifiedheparinase III enzyme having a substitution of an alanine for histidineat amino acid residue 225 (MO11) in aqueous sodium acetate buffer, pH7.2 at 37° C. to produce Intermediate 2. MO11 cleaved by υ-eliminationbetween N-acetylglucosamine residues and under sulfated uronic acidsproducing chains having a Δ4,5 uronic acid group at the non-reducing endand an N-acetyl glucosamine at the reducing end. When digestion wascomplete, heat was turned off and sodium chloride was added to achieve afinal solution concentration of approximately 2% w/v.

In Step 3, size exclusion chromatography (SEC) was used to separate thehigh anti-factor Xa and IIa components of Intermediate 2 away from thelower activity materials. The product of this step was designatedIntermediate 3.

In Step 4, individual or combined Intermediate 3 materials weredissolved in purified water, filtered through a 0.2 pm filter, andlyophilized to produce M118-REH drug substance.

Starting Material in the Manufacture of M118-REH

Specifications of Starting Materials

The starting material for making M118-REH, UFH sodium (USP) is ofporcine intestinal mucosa origin. In addition to the USP tests,additional controls have been put in place. These controls are listed inTable 1:

TABLE 1 UFH Assays and Specifications in Addition to DMF TestSpecification Certificate of Analysis Potency NLT 160 U/mg on a driedbasis Agarose Gel Electrophoresis Report Fast-Moving and Slow-Movingheparin Report Dermatan sulfate and Chondroitin sulfate

The Agarose Gel Electrophoresis (AGE) semi-quantitatively separates thevarious components of heparin-based materials, as dermatan sulfate andchondroitan sulfate on the basis of their electrophoretic mobility. Ahorizontal separation in 0.5% agarose gel was conducted in bariumacetate buffer pH 5.8, followed by 1,3-diaminopropane acetate pH 9buffer. A special electrophoresis tank was employed, whereby theelectrode chambers containing liquid buffer were overlaid with awater-immiscible, low density organic solvent (e.g., petroleum ether orheptane). This design provided efficient heat transfer between theagarose gel plate and a metal cooling tray filled with ice.

Low molecular weight heparins traditionally are prepared from USP gradeUFH. To achieve a higher potency low molecular weight heparin drugsubstance, the UFH starting material for the M118-REH process wasrestricted to those with potencies in excess of 160 IU/mg. To controlthe levels of dermatan sulfate and chondroitin sulfate, these weremeasured in the UFH starting material and controlled in Step 1 of theM118-REH manufacturing process.

Structural Analysis of Intermediates

The steps in the M118-REH manufacturing process are outlined above andshown in FIG. 1. Characterization technology for studying sugarstructure provides an understanding of the structural attributes thatchange when UFH is subjected to this process at each step. Thisinformation allows control and reproducibility from the process,including selection of starting material.

Analysis of different Step 2 materials (or Intermediate 1 samples) andthe starting UFH samples used to prepare them was carried out using2D-NMR(HSQC) analysis, and capillary electrophoresis.

The building blocks that constitute the starting material as well as theintermediate were identified and quantified. Some of the Step 2materials studied were not ideal substrates for the next step (enzymaticdigestion) and attributes were identified that indicate preferred step 2material.

Shown in Table 2 and Table 3 below are data from 2D NMR analysis thatillustrate the differences between the starting UFH and step 2 material.This analysis allows determination of the overall differences instructural attributes when going through Step 1 of the M118-REHmanufacturing process. Based on all the data obtained from the differentanalyses certain conclusions about the starting material, intermediatesand manufacturing process were made.

TABLE 2 2D NMR analysis of UFH and the step 2 materials obtained fromthem. These materials represent the preferred step 2 material. Step 2Step 2 Monosaccharide UFH #1 #1 #2 UFH #2 #3 Glucosamine H_(NS)-(I_(2S))63.7 57 57.1 65.6 56.3 H_(NS)-(I) 9 11.1 12.3 7.4 12.3 H_(NS)-(G) 7.712.7 11.2 8.9 11.8 H_(NAc (internal)) 12.1 13.9 14.6 11.1 13.3H_(NS, 3S) 6.1 4.4 4.8 7 5.5 H_(6S) 78.2 85.3 85.8 82.5 86.7 LinkageRegion 4.2 3.6 3.8 2.1 1.8 (L.R.) ΔU 0 0 0 0 0 I_(2S) 73 66.8 70.9 75.970.2 I-(H_(NS/Ac, 6S)) 8.1 11.8 9.4 7.2 8.5 I-(H_(NS/Ac)) 1.4 2 1.4 0.81.1 G-(H_(NS)) 8.1 11.2 8.4 8 9.6 G-(H_(NS, 3S)) 2.8 2.8 3.2 2.7 3.7G-(H_(NAc)) 6.5 5.3 6.6 5.3 4.6 Epoxide 0 0 0 0 0.6

The step 2 materials had lower relative I_(2s) content when compared tostarting material and this was also reflected by the decrease inH_(NS)-(I_(2S)) structure as shown in the table. This was accompanied bya concomitant increase in the H_(NS)-(I) and H_(NS)-(G) structures asexpected. Interestingly, there was also a relative increase in theamount of 6-O-sulfated hexosamine (H_(6S)).

TABLE 3 2D NMR analysis of UFH and the step 2 materials obtained fromthem. These materials represent the less preferred step 2 material. Step2 Step 2 Monosaccharide UFH #1 #1 UFH #2 #2 Glucosamine H_(NS)-(I_(2S))68.1 63 66.6 61.8 H_(NS)-(I) 6.3 10.7 8.6 11.9 H_(NS)-(G) 8.5 12.7 9.511.1 H_(NAc (internal)) 11.2 8.3 9.8 8.1 H_(NS,3S) 5.9 5.3 5.4 6.8H_(6S) 82.2 89.2 84.7 88.4 Linkage Region 2.1 1.5 1.8 1.1 (L.R.) ΔU 0 00 0 I_(2S) 76.8 71.3 76.1 69.8 I-(H_(NS/Ac,6S)) 6.7 6.3 6.2 7.1I-(H_(NS/Ac)) 0.6 0.8 2 1.6 G-(H_(NS)) 7.2 10 6.6 10.3 G-(H_(NS,3S)) 1.62.3 3.7 3 G-(H_(NAc)) 4.3 2.4 3.6 2.1 Epoxide 2.8 3.6 1.8 2.4

“Preferred” step 2 materials are those that are good substrates for thenext step in the process i.e. enzymatic digestion by MO11, whereas “lesspreferred” step 2 materials are poorer substrates. When comparing therelative amounts of the H_(NAc) (internal) it was observed that in“preferred” step 2 materials the H_(NAc) content actually goes up afterstep 1 whereas in “less preferred” step 2 materials it is reduced (seeTable 2 and 3). Another observation was that the relative amount of theG-H_(NAc) unit was reduced to a much larger extent in “less preferred”step 2 as compared to “preferred” step 2 material. These observationswere justified in the context of substrate specificity of MO11 whichprefers to act on the linkage adjacent to non-sulfated glucuronic acid(i.e. H_(NAc)-G).

Through the analysis, structural attributes that change during the firststep (precipitation) of the manufacturing process going from UFH to step2 material were identified. Since the N-acetyl content in the step 2material appears to be important for the subsequent enzymatic digestionstep, it is desirable to use a starting UFH with a higher N-acetylcontent which may allow better production of step 2 material afterprecipitation. Therefore, based on this analysis, a preselectedcriterion for starting UFH has been identified that allows bettercontrol and evaluation of the M118-REH manufacturing process.

Other assays and specifications for process intermediates are shown inTable 4, and described in detail below.

TABLE 4 Assays and Specifications for Intermediates Intermediate AssaySpecifications 1 Agarose Gel Electrophoresis: Dermatan and Dermatan andChondroitin Chondroitin Sulfate Sulfate below detection limit belowdetection limit 2 Automated Chromogenic Assay 2 SEC-MALS 3 AutomatedChromogenic Assay Anti-Factor Xa NLT 130 IU/mg Automated ChromogenicAssay SEC-MALS Molar Mass: 5000-9000 Dalton Polydispersity (PD): NMT 1.5

The Agarose Gel Electrophoresis process has been described above.

The anti-factor Xa activity was measured as described herein. Theanti-factor Xa activity was measured on either a Diagnostica Stagoanalyzer with the Stachrom® Heparin Test kit, or on an ACL Futura3Coagulation system with the Coatest® Heparin Kit from Chromogenix. TheAnalyzer response was calibrated using the NIBSC International Standardfor Low Molecular Weight Heparin, lot 01/608 or current lot. The potencyof M118-REH Drug Substance was calculated in International Units ofanti-factor Xa activity per milligram using the statistical methods forparallel line assays.

The anti-factor IIa activity was measured as described herein. using thefollowing principle. The anti-factor IIa activity was measured on eithera Diagnostica Stago analyzer or on an ACL Futura3 Coagulation system,with reagents from Chromogenix (S-2238 substrate, thrombin (53nkat/vial), and antithrombin). The Analyzer response was calibratedusing the 2nd International Standard for Low Molecular Weight Heparin,lot 01/608 or equivalent. The potency of M118-REH Drug Substance wascalculated in International Units of anti-factor IIa activity permilligram using the statistical methods for parallel line assays.

The weight average molar mass, the polydispersity and the molar massdistribution of M118-REH Intermediates were measured using a SizeExclusion Chromatography (SEC) system attached to a Wyatt miniDAWN MultiAngle Light Scattering (MALS) detector or any other suitable MALSdetector, and an Optilab rEX interferometric refractometer (RID) orother suitable RID in accordance with the USP <621>, current version.The SEC columns set consisted in columns packed with a high resolutionL20 packing, for example a Tosoh SWXL guard column coupled with a TosohTSKgel G3000SWXL and a Tosoh TSKgel G2000SWXL in series. The system wasequilibrated at 0.5 mL/min with a 0.2M sodium sulfate mobile phase whosepH was adjusted to 5.0 with sulfuric acid. Sodium azide was added at0.05% in the mobile phase. The M118-REH Intermediate was dissolved inthe mobile phase to obtain a 10 mg/mL solution prior to injection. Theweight average molar mass, the polydispersity and the distributionparameters were measured using the Wyatt Astra software or anyappropriate software. The distribution was characterized by thepercentage of chains with a molar mass lower than 5,500 Da (M₅₅₀₀), andthe percentage of chains with a molar mass higher than 8,000 Da (M₈₀₀₀)or by the percentage of chains with a molar mass lower than 5,000 Da(M₅₅₀₀), and the percentage of chains with a molar mass higher than7,500 Da (M₈₀₀₀).

In Step 2 of the M118-REH process, Intermediate 1 was digested with theMO11 enzyme. As the enzyme digested the Intermediate 1 substrate, itgenerated Intermediate 2 containing Δ4,5 uronic acid residues possessinga characteristic UV₂₃₂ absorbance. To monitor the progress of thedigestion, the reaction solution was sampled periodically and theabsorbance at 232 nm was measured. The Step 2 digestion was consideredcomplete when the absorbance at 232 nm has not changed more than 2 AU in1 hour.

Table 5 shows the weight average molecular weight and distribution ofvarious preparations of M118-REH. Table 6 shows a comparison ofM118-REH, LMW and UFH products.

TABLE 5 Molecular weight, polydispersity and chain lengthcharacteristics of 5 different lots of M118-REH LOT # Mw (Da) PD M5500M8000 n 1 7250 1.1 24.6% 31.9% 13 2 7300 1.1 26.4% 33.0% 13 3 7500 1.124.6% 35.0% 13 4 6350 1.1 38.6% 17.7% 12 5 6450 1.1 40.6% 20.7% 12 * MWcalculated with dn/dc measured on M118-REH material.

TABLE 6 Comparison of M118, LMWH and UFH Products Attribute M118-REH¹Lovenox² UFH³ Anti-Xa Activity (IU/mg) 228 100 150 Anti-IIa Activity(IU/mg) 155 25 150 Anti-Xa/Anti IIa Ratio 1.5:1 4:1 1:1 AverageMolecular Weight 6350 4,500 12,000 Polydispersity 1.1 1.3 1.6Subcutaneous bioavailability Yes Yes No Reversibility w/protamine FullPartial Full Monitorable with ACT/APTT Yes No Yes ^(1,)* Values for lotused in M118-REH Drug Product formulation; average MW processed withdn/dc estimated from literature data on UFH ²Lovenox package insert ³USPheparin monograph

Structural Characterization of M118-REH: Identification of StructuralCharacteristics of M118-REH

An approach towards the characterization of M118-REH has been developedthat involves several different analytical techniques that providecomplementary sets of data.

This provides characterization of M118-REH, and it allows for anunderstanding of what makes M118-REH unique when compared to otherLMWHs. The summaries of findings from these characterization techniquesthat help define M118-REH as a unique mixture.

A characterization of unfractionated heparin (UFH) and LMWH products wascompleted using a series of analytical techniques that has led to theidentification of chemical structures unique to a given LMWH, structuresthat are present in several different LMWHs but at varying amounts, andstructures that are responsible for the biological properties ofheparins.

Analysis of a complex LMWH mixture like M118-REH needs to account fornot only the inherent structural variability that arises from thebiosynthesis of heparin, but also for the structures that arise from theenzymatic cleavage and manufacturing processes. This can be addressed byresolving the natural as well as modified (if any) reducing andnon-reducing end signatures present in the mixture. At the same time, italso needs to be confirmed that the relative “order” of the disaccharideunits, as defined by the parent UFH molecule, is not affected by themanufacturing process. Therefore, it is necessary to provide a sequencecontext in which these modified or natural building blocks are presentin the chains of M118-REH. To account for these factors, an approachtowards the characterization of M118-REH has been developed thatinvolves using data obtained from different analytical techniques thatprovide unique and complementary sets of data.

The composition analysis was performed with CE to identify and quantifyindividual building blocks that comprise the M118-REH chains. Thesemethods also identify the building block structure that is responsiblefor anti-Xa activity, referred to as “Anti-Xa Building Block”.

Building Block Analysis/Compositional Analysis

Compositional Analysis by Capillary Electrophoresis (CE)

Briefly, this method involves the enzymatic digestion of M118-REH intoits constituent building blocks followed by separation using CE (FIGS.2A and 2B).

CE is a high resolution separation technique and has been usedextensively in the analysis of UFH and other glycosaminoglycans. Thecurrent method used capillary zone electrophoresis in an uncoated fusedsilica capillary. With capillary electrophoresis, the most highlysulfated species migrated through the capillary the fastest and aredetected first.

Representative Data

The profile of enzyme digest for M118-REH as observed by CE is shown inFIG. 2. Notably, no modified building blocks beyond those alreadypresent in UFH were observed in M118-REH (indicated by the 1,6structures observed for enoxaparin). Another interesting observation wasthat the amount of 3-O-sulfated species was almost doubled in M118-REHas compared to enoxaparin (indicated by the AT-III tetra peak). Thiscorrelated well with the higher anti-Xa activity observed for thismixture and was consistent with the methodology for production ofM118-REH. The extent of overall sulfation was also shown to be slightlyhigher for M118-REH than enoxaparin based on this technique.

This technique was also used to determine the presence and quantity ofeach of the building block saccharide components of M118-REH (Table 12).

TABLE 7 Building block saccharides observed in CE analysis of M118-REHPeak Structure 1 ΔU_(2S)H_(NS,6S) 2 ΔU_(2S)H_(NS) 3 ΔUH_(NS,6S) 4ΔU_(2S)H_(NAc,6S) 5 ΔUH_(NS) 6 ΔU_(2S)H_(NAc) 7 ΔUH_(NAc,6S) 8 ΔUH_(NAc)9 ΔUH_(NAc,6S)GH_(NS,3S) 10 ΔUH_(NAc,6S)GH_(NS,3S,6S) 11ΔU_(2S)H_(NS,6S)I_(2S) 12 ΔU_(2S)H_(NS,6S)GH_(NS,3S,6S) 13ΔU_(gal)H_(NS,6S) 14 ΔU_(gal)H_(NS)

Qualitatively, no 1,6-anhydro building blocks (observed in enoxaparin)or 2,5-anhydro structures (seen in dalteparin) were observed in theM118-REH profile. Some interesting observations arise from thequantitative analysis of these structures. When comparing the relativemole % of peak 10 (ΔUH_(NAc,6S)GH_(NS,3S,6S)) between enoxaparin andM118-REH, a higher amount of the peak was present in M118-REH (Table 6),confirming that the M118-REH manufacturing process enriched for theactive anticoagulant sequences in heparin. The amount of trisaccharide(ΔU_(2S)H_(NS,6S)I_(2S)) was relatively low in M118-REH as compared toenoxaparin. This is a reflection of the process for manufacture. Thechemical process used to make enoxaparin results in “peeling” from thereducing end of oligosaccharides, thereby increasing the number of oddnumbered chains. This is not the case for M118-REH and so the level oftrisaccharide was a direct consequence of what was observed in thestarting UFH. This analysis indicates that the CE methodology wassensitive enough to actually pick up these changes that are indicativeof different processes used for manufacturing LMWHs and so it can bevery discriminatory.

TABLE 8 Quantitative comparison of selected structures: M118-REH andenoxaparin Structure M118 Enoxaparin ΔUH_(NAc,6S)GH_(NS,3S,6S) 8.6 4.7ΔU_(2S)H_(NS,6S)I_(2S) 0.6 1.9

Compositional Analysis by 2D NMR

NMR spectroscopy has been successfully used for detecting andquantifying signals associated with major or minor structural featuresin polysaccharides. NMR spectroscopy is also one of the only techniquesthat allow an effective determination of the iduronic and glucuronicacid components in the mixture. Two dimensional (2D)NMR spectroscopy hasbeen used as a means of resolving and identifying distinct signals thatcorrespond to a certain population of monosaccharide residues. Thisapproach enables one to not only quantify the basic monosaccharideconstituents of the mixture, but to also assess their linkageenvironments in a quantitative manner.

Two dimensional NMR provides a complementary technique to thecompositional analysis of M118-REH by CE. 2D NMR provides information onH-U linked disaccharides, thereby providing complementary analysis ofdisaccharide linkages. Recent methodology using 2D proton-carboncorrelation spectroscopy (HSQC) experiments has demonstrated the abilityto obtain this quantitative compositional analysis onglycosaminoglycans.

Spectra of the anomeric region of M118-REH, as measured using 2Dproton-carbon correlation spectroscopy (HSQC) are presented in FIG. 4.The cross peaks in the anomeric region are shown in the Figure.

An analysis of the anomeric region of M118-REH provided some veryinteresting information regarding what makes M118-REH unique compared toother LMWHs. First, the anomeric region is much simpler when compared toother LMWHs, like enoxaparin. Second, when analyzing the reducing endresidues of the chains, it was observed that a majority of the chainsend in N-acetylglucosamine, and only a minor amount of the chains end inN-sulfoglucosamine. This arises as a result of the specificity of theenzyme used to prepare M118-REH. Third, the NMR data indicate that ˜30%of the chains have a ΔU residue at the non-reducing end, which is,again, a result of the enzyme specificity. Fourth, no G-H_(NAc)disaccharide was observed in M118-REH. Finally, no linkage regionsaccharide was observed in the NMR spectrum. The percentage compositionof monosaccharides in M118-REH and their linkage environments arereported in Table 7. NMR analysis also enables determination of theiduronic acid/glucuronic acid ratio for M118-REH.

TABLE 9 Percentage composition of glucosamine and uronic acid residuesin M-118 (results of two experiments) M118-REH GlucosamineH_(NS)-(I_(2S)) 57.6/57.0 H_(NS)-(I)  9.8/12.0 H_(NS)-(G) 11.0/11.1H_(NAc (internal)) 6.1/3.1 H_(NS,3S) 7.3/7.2 H_(NS)red 1.6/1.5H_(NAc)αredox 4.6/5.1 H_(NAc)βredox 2.0/3.0 H_(6S) 90.4/90.5 L.R.    0/<0.1 Uronates ΔU 1.9/2.4 I_(2S) 68.7/70.2 I-(H_(NS/Ac,6S)) 9.9/9.5I-(H_(NS/Ac)) 1.4/0.9 G-(H_(NS)) 8.7/7.7 G-(H_(NS,3S)) 5.9/4.8G-(H_(NAc)) 0/0 GalA 2.7 Epox 0.8

Conclusions

The following were identified as structural attributes of M118-REH:

-   -   Enrichment of 3-O-sulfate containing saccharide chains (AT-III        binding tetrasaccharide) based on relative mole % as compared to        existing LMWHs and the UFH starting material;    -   Generation of a predominant reducing end structure (H_(NAc));    -   The only modified non-reducing end structure observed is ΔU;    -   Maintenance of the natural disaccharide backbone structure of        UFH with limited introduction of process-related changes that        are observed in other LMWHs;    -   Removal of UFH components from the mixture that are not required        for anti-Xa or anti-IIa (thrombin) binding, thereby creating a        more defined heparin;    -   The specificity of the M118-REH depolymerization process retains        the required chain length and proximal separation of the binding        sites in order to retain anti-IIa activity.

A key attribute of all LMWH products is that longer polysaccharidechains are cleaved into smaller fragments via a variety ofdepolymerization methods, as depicted in FIG. 5. This cleavage event,which can be caused by either chemical or enzymatic reactions, resultsin characteristic signatures at both the reducing and non-reducing endsof the molecule. The characteristic end groups present is M118-REH aredescribed below.

Structure at the Non-Reducing End

The specificity of the enzymatic cleavage that resulted in the formationof a new non-reducing end (abbreviated as ΔU), enabled the positioningof the AT-III binding site within the polysaccharide chain.

Structure at the Reducing End

The glucosamine structures at the new reducing ends on the M118-REHchains were reflective of the specificity of the enzyme used in theprocess. As a result, the majority of the reducing end structures inM118-REH were N-acetylated hexosamine (H_(NAc)) residues.

Structural Differences Between M118-REH and Unfractionated Heparin

-   -   1) M118-REH has a higher mole % of the antithrombin binding        saccharide sequence as compared to the starting unfractionated        heparin (UFH).    -   2) M118-REH has negligible amount of linkage region as compared        to the starting UFH.    -   3) M118-REH has a certain percentage on Δ4,5 glucuronic acid at        the non-reducing end of chains, whereas UFH does not contain        this modified residue at the non-reducing end.

In summary, M118-REH is a heparin product having unique physical andfunctional attributes.

Most of the structural attributes discussed above were a directconsequence of the properties of the enzyme used to prepare M118-REH.These include the predominant reducing end structure (H_(NAc)), the onlymodified non-reducing end structure (ΔU) as well as the removal oflinkage region. Also since the enzyme preferentially cleaves the loweror non-sulfated domains in the heparin mixture, it does not affect theAT-III binding sequence, which, as a result, is enriched in M118-REH

The characterization protocol defined above was used to analyze 4batches of M118-REH. This analysis confirms consistency in manufactureof what is defined as M118-REH.

TABLE 10 CE analysis of several batches of M118-REH Peak Structure #1 #2#3 #4 1 ΔU_(2S)H_(NS, 6S) 57.1 59.5 57.0 57.4 2 ΔU_(2S)H_(NS) 6.0 5.65.4 6.1 3 ΔUH_(NS, 6S) 13.2 12.3 12.2 12.7 4 ΔU_(2S)H_(NAc, 6S) 1.8 1.61.7 1.8 5 ΔUH_(NS) 0.6 0.7 0.6 0.7 6 ΔU_(2S)H_(NAc) 0.5 0.5 0.4 0.4 7ΔUH_(NAc, 6S) 4.5 3.7 3.7 4.3 8 -UH_(NAc) 1.8 1.3 1.5 1.8 9ΔUH_(NAc, 6S)GH_(NS, 3S) 1.3 1.0 1.2 1.2 10 ΔUH_(NAc, 6S)GH_(NS, 3S, 6S)10.4 9.4 8.9 8.6 11 ΔU_(2S)H_(NS, 6S)I_(2S) 0.3 0.3 0.7 0.6 12ΔU_(2S)H_(NS, 6S)GH_(NS, 3S, 6S) 0.5 0.7 1.0 0.9 13 ΔU_(gal)H_(NS, 6S)1.7 2.9 4.4 3.0 14 ΔU_(gal)H_(NS) 0.2 0.5 0.9 0.4

TABLE 10A Preferred ranges for peaks Peak Structure A 1 ΔU_(2S)H_(NS,6S)58 ± 5  2 ΔU_(2S)H_(NS)   6 ± 2.5 3 ΔUH_(NS,6S) 13 ± 3  4ΔU_(2S)H_(NAc,6S) 1.5 ± 1.5 5 ΔUH_(NS) 0.6 ± 1.0 6 ΔU_(2S)H_(NAc) 0.5 ±1.0 7 ΔUH_(NAc,6S) 4 ± 2 8 ΔUH_(NAc) 1.5 ± 2.0 9 ΔUH_(NAc,6S)GH_(NS,3S)1.2 ± 1.5 10 ΔUH_(NAc,6S)GH_(NS,3S,6S) 9.5 ± 4   11ΔU_(2S)H_(NS,6S)I_(2S) 0.5 ± 1.0 12 ΔU_(2S)H_(NS,6S)GH_(NS,3S,6S) 0.7 ±2.0 13 ΔU_(gal)H_(NS,6S) 3.0 ± 3.0 14 ΔU_(gal)H_(NS) 0.6 ± 1.5

TABLE 11 Percent composition of M118-REH glucosamine and uronic acidresidues Monosaccharide 1 2 3 4 Glucosamine H_(NS)-(I_(2S)) 54.3 57.061.8 57.6 H_(NS)-(I) 12.5 12.0 11.0 9.8 H_(NS)-(G) 10.8 11.1 9.5 11.0H_(NAc (internal)) 3.4 3.1 4.2 6.1 H_(NS,3S) 8.7 7.2 7.0 7.3 H_(NS)red0.8 1.5 0.4 1.6 H_(NAc)αred 6.0 5.1 4.1 4.6 H_(NAc)βred 3.6 3.0 2.0 2.0H_(6S) 90.0 90.5 91.3 90.4 Linkage Region <0.1 <0.1 <0.1 <0.1 UronatesΔU 2.7 2.4 2.1 1.9 I2S 69.8 70.2 70.6 68.7 I-(H_(NS/Ac,6S)) 11.8 9.5 9.29.9 I-(H_(NS/Ac)) 1.4 0.9 1.2 1.4 G-(H_(NS)) 8.9 7.7 8.4 8.7G-(H_(NS,3S)) 5.4 4.8 4.1 5.9 G-(H_(NAc)) 0 0 0 0 Galacturonic Acid 02.1 2.4 2.7 Epoxide 0 2.4 2.0 0.8

TABLE 11A Preferred ranges for structures Monosaccharide A GlucosamineH_(NS)-(I_(2S)) 57.7 ± 7  H_(NS)-(I) 11.3 ± 5  H_(NS)-(G) 10.6 ± 5 H_(NAc (internal)) 4.2 ± 5 H_(NS,3S) 7.6 ± 5 H_(NS)red 1.1 ± 5H_(NAc)αred 5.0 ± 5 H_(NAc)βred 2.7 ± 5 H_(6S) 90.6 ± 6  Linkage Region <0.1-0.0 Uronates ΔU 2.3 ± 5 I2S 69.8 ± 6  I-(H_(NS/Ac,6S)) 10.1 ± 6 I-(H_(NS/Ac)) 1.2 ± 5 G-(H_(NS)) 8.4 ± 5 G-(H_(NS,3S)) 5.1 ± 5G-(H_(NAc))   0 + 2 Galacturonic Acid 1.8 ± 5 Epoxide 1.3 ± 5

Description and Composition of the Drug Product M118-REH Injection

The drug product, M118-REH Injection, is a clear, colorless to slightlyyellow solution in a 3 mL single use, Type 1 glass vial, sealed with achlorobutyl stopper and oversealed with an aluminum crimp. Each vialnominally contains 5000 IU of anti-factor Xa activity in 2 mL.

The quantitative composition of M118-REH Injection is given in Table 11.The composition is given for the labeled volume of 2 mL. The vials werefilled with 2.15 mL, consistent with the USP recommended excess volume.

TABLE 12 Composition of M118-REH Injection Amount per unit QualityComponent (Vial) Function Standard M118-REH Drug 5000 IU anti-Xa ActiveN/A Substance activity¹ Pharmaceutical Ingredient Sodium ChlorideIn-process Osmolality Agent USP OSmolality Adjustment² Water forInjection q.s. to 2 mL Solvent USP ¹The amount of M118-REH drugsubstance is calculated based on the anti-factor Xa activity (on a driedbasis) and the Loss on Drying. Assuming anti-factor Xa activity of 200IU/mg, the quantity of M118-REH drug substance is 25 mg per vial. ²Thequantity of Sodium Chloride required to achieve an osmolality of 280-330mOsm/L is approximately 8 mg/mL, or 16 mg per vial.

No diluent was required for use with M118-REH Injection.

Components of M118-REH Injection

M118-REH Injection was manufactured by dissolving M118-REH DrugSubstance in Water for Injection. M118-REH Drug Substance is verysoluble in aqueous solution and the particle size distribution of drugsubstance therefore had no effect on the performance of the drugproduct.

Sodium Chloride, USP was the only excipient used in M118-REH Injection(at a concentration of approximately 8 bg/mL). Sodium chloride wasselected as an osmolality adjusting agent to avoid injection sitediscomfort and haemolysis upon administration.

Manufacturing Process Development of M118-REH Injection

The M118-REH Injection manufacturing process consisted of dissolving theM118-REH drug substance in Water for Injection, USP, and adjusting theosmolality with Sodium chloride, USP. The formulated solution wasfiltered through two 0.2 pm filters in series and aseptically filledinto vials. Heparin sodium products are subject to degradation at veryhigh temperatures and therefore cannot be terminally sterilized. Theprocess flow diagram for M118-REH Injection is presented in FIG. 6(described below).

FIG. 6 shows a process flow diagram for M118-REH Injection. The amountof M118-REH drug substance to be added to each batch was calculatedbased on the Assay (anti-factor Xa activity) and Loss on Drying valuesfrom the Certificate of Analysis according to the following calculation:

2500 IU/mL/Assay (IU/mg)×100/(100−Loss on Drying %)×Batch size (mL)÷1000mg/g=Quantity of Drug Substance to add (g)

Water for Injection equivalent to approximately 75% of the final batchweight was added to the formulation vessel and mixing was initiated. Thecalculated amount of M118-REH drug substance was slowly added to thevessel and mixed until all solid was dissolved. An initial quantity ofSodium Chloride USP was added and the solution was mixed until all solidwas dissolved. Water for Injection was added to the final batch weightand the solution was mixed for an additional 5-15 minutes. Theosmolality was measured, and additional Sodium Chloride USP was added,if required, to achieve an osmolality of 280-330 mOsm/L.

Two pre-sterilized Millipak 20 PVDF (polyvinylidene fluoride) 0.22 Tmfilters in series were used to sterilize the M118-REH bulk drug product.The product was filtered into a filling reservoir, and, at the end offiltration, the filters were integrity tested.

Biological and Pharmacological Properties of M118-REH

M118-REH is the product of an enzymatic digest that results in adepolymerized low molecular weight heparin. The depolymerized pool isenriched for active anticoagulation and antithrombotic fractions, whichmay be a consequence of the site specific digestion by a specificglycosaminoglycan lyase. The specific site and orientationdepolymerization via this enzyme has enabled M118-REH to be a highlyefficacious molecule on artery injury protection. Furthermore, M118-REHhas the attribute of being reversible and easily monitored by bedsideclotting assays.

Studies of M118-REH have been done to reveal the pharmacologic andbiologic properties of M118-REH, and through this process, its mode ofactions have been explored both in vitro and in vivo. The mechanism ofanti-coagulation and anti-thrombotic function have been investigated.The preliminary Structure and Activity Relationship (SAR) has beenaddressed in these studies. These studies and their results aredescribed as the following, which are initial analyses for generatingthe profile of M118-REH's biopharmacologic activities.

In Vitro Analysis of Coagulation Activity:

In vitro anti-Xa activity of M118-REH ranges from 180-300 IU/mg based on2nd international low molecular weight heparin standards. M118-REHpreparations higher in vitro anti-Xa/IIa activities are proportional toits fraction as ΔUH_(Nac,6s)GH_(NS,3S,6S) containing 3-O-sulfationmoiety.

In vitro anti-IIa activity of M118-REH ranges from 100-250 IU/mg basedon 2^(nd) international low molecular weight heparin standards.

M118-REH can prolong the aPTT in vitro from 40 sec to 80 sec. at 2.4μg/ml and aPTT change is propositional to the anti-Xa and anti-IIaactivity.

In Vitro Neutralization by Protamine Sulfate and Measured by Anti-XaActivity:

Protamine can fully reverse the anti-Xa activity of M118-REH at ratio of1 mg:100 anti-Xa IU in human plasma. This can be compared to other LMWHpreparations. For example, protamine can only neutralize 60% anti-Xaactivity of enoxaparin at ratio of 3 mg:100 anti-Xa IU in human plasma.These results are shown in FIG. 7.

In Vitro Human Umbilical Vein Endothelial Cells (HUVECs) Release TissueFactor Pathway Inhibitor (TFPI):

HUVECs from ATCC were grown in 2% FBS F12K modified medium without ECGS.M118-REH, Lovenox, and UFH were prepared in the same medium with thefinal concentration of 0.01 mg/ml and 0.005 mg/ml. Three wells of cellsfor each group were incubated under 37° C., 5% CO₂, and 95% O₂ for 24and 48 hours. The supernatant was taken out for TFPI release test usingELISA kit from ADI.

M118-REH at 0.005 mg/ml and 0.01 mg/ml significantly increased the TFPIrelease from HUVECs at 24 and 48 hours and, as shown in FIG. 8. M118-REHresulted in more release of TFPI from HUVECs into cell medium thanunfractionated heparin. Lovenox did not cause a significantly higherTFPI release when compared with control.

Pharmacokinetics of M118-REH In Vivo:

In rodent models such as Sprague-Dawley rats and B16B16 mice, M118-REHhas longer elimination half life than UFH and comparable to that ofenoxaparin after intravenous injection. M118-REH is quickly absorbedafter subcutaneous injection with Tmax ranged from 1 hour to 3 hours.

In rabbit model such New Zealand white rabbit, M118-REH subcutaneousinjection yields higher bioavailability in terms of both anti-Xa andanti-IIa activity than that of UFH, the bioavailability ranged from 50%to 100% compared with intravenous injection. The pharmacokinetics ofM118-REH is comparable to that of enoxaparin with elimination half liferanged from 3-5 hours and the major elimination mechanism is throughrenal excretion.

TABLE 13 Pharmacokinetics parameters of M118-REH, enoxaparin and UFHafter intravenous injection in a rabbit and rat model. M118-REHEnoxaparin UFH T½ (hr) Anti-Xa 0.87 ± 0.25 1.93 ± 0.45 0.54 ± 0.03Rabbit i.v activity 1.5 mg/kg Anti-IIa 0.85 ± 0.05 1.78 ± 0.63 0.89 ±0.05 activity T1/s (hr) 1 mg/kg 0.39 ± 0.06 0.41 ± 0.07 0.35 ± 0.06 Rati.v 0.5 mg/kg 0.29 ± 0.12 N/A 0.19 ± 0.04 Anti-Xa

In NHP, such as Cytomologus monkey, the elimination half life ofM118-REH ranged from 20 minutes to 50 minutes. The anti-Xa/IIa ratioafter intravenous injection was kept consistent during the PK coursewhich ranged from 0.5 to 2 and M118-REH was distributed in thecirculation system and eliminated through renal route based on theanalysis of pharmacokinetic parameters. Table 14 depictspharmacokinetics of M118-REH in NHP model.

TABLE 14 Pharmacokinetics of M118-REH in NHP. Test Dose t½ CmaxAUCINF_obs Vz_obs Cl_obs material Group (IU/kg) Rsq (hr) (IU/mL)(hr*IU/Ml) (mL/kg) (mL/hr/kg) Anti-Xa 1 Mean 150.0 0.98 0.50 4.07 4.0727.26 37.24 SD 0.9 0.02 0.04 0.15 0.54 5.57 4.65 Anti-IIa 2 Mean 150 1.00.68 2.733 3.45 43.17 43.92 SD 0.0 0.01 0.05 0.38 0.45 3.66 5.38 Thepharmacokinetics of M118-REH represents the first order of elimination.

In vivo TFPI concentration remained at high levels over 24 hours afterM118-REH dosing. ACT and aPTT both correlated very well with the anti-Xaactivity.

Pharmacodynamics Study of M118-REH In Vivo:

In rodent models such as Sprague-Dawley rat, M118-REH and UFH wereintravenously injected at 1 or 2 mg/kg via jugular vein, whileenoxaparin was dosed at 0.5 or 1 mg/kg. ACT (activated clotting time)was measured with Hemochron Jr. In this model, activated partialthromboplastin time (aPTT) and activated clotting time presented doseresponse to M118-REH escalating dosages. ACT increased 1.5-3 folds afterM118-REH delivery at 0.5 mg/kg while 2-4 folds at 1 mg/kg afterintravenous injection. The pharmacodynamic profile was similar to thatof the pharmacokinetics in terms of anti-Xa/IIa measurement; theelimination half life ranges from 0.15 to 0.5 hour.

In a rabbit model such as New Zealand white rabbit, aPTT and ACT weremeasured after M118-REH delivery intravenously. aPTT and ACT increasedproportional to M118-REH dose. The anti-Xa/IIa ratios were consistentafter both intravenous and subcutaneous administration at 1.5 mg/kg. Incontrast to those of M118-REH, the anti-Xa/IIa ratios of both enoxaparinand UFH after intravenous administration fluctuated significantly duringthe course.

As shown in FIG. 9, in NHP models such as Cynomologus monkey, theresults showed that aPTT and ACT increased significantly 2-4 folds and1.5-3 folds after M118-REH intravenous injection at 150 anti-Xa IU/kg.

M118-REH intravenous bolus injection enhanced TFPI release into theblood stream by 2-20 folds and such effects last more than 24 hours.

The effects of M118-REH intravenous bolus injection followed bycontinuous infusion were also studied in a canine model of deep arterialthrombosis induced by severe electrolytic injury. More details regardingthis study are provided below in the Example entitled “Beagleelectrolytic-induced femoral artery injury model”.

Efficacy Study of M118-REH in Preclinical Models:

Ferric Chloride-Induced Carotid Artery Injury

Comparison studies of M118-REH and enoxaparin yielded dose-dependentinhibition of occlusive thrombosis. M118-REH at a dose of 0.5 mg/kgsignificantly (p<0.05) prolonged the time to occlusion (TTO) compared tosaline (28.5±7.1 minutes versus 11.1±0.9 minutes, respectively).Twenty-five percent (2/8) of the injured carotid artery injected with0.5 mg/kg M118-REH remained patent for the entire 60 minute observationperiod. In contrast, all (9/9) of the injured carotid arteries occludedin rats injected with saline within the 60 minute observation period.Administration of 1 mg/kg of M118-REH further increased TTO (55.3±3.6minutes) with 83% (10/12) of the vessels patent at the end of the 60minutes observation period. Rats administered 2, 3 or 4 mg/kg ofenoxaparin all had significantly longer TTO than animals injected withsaline (19.1±1.4, 36.0±5.6 and 40.2±6.3 minutes, respectively). All(7/7) of the carotid arteries occluded within the 60 minute observationperiod at the 2 mg/kg dose while 62% (7/13) and 55% (6/11) vesselsoccluded in rats administered with 3 and 4 mg/kg enoxaparin,respectively. M118-REH (1 mg/kg) produced the greatest degree ofprotection from thrombosis in spite of lower anti-Xa activity than thatat the 3 and 4 mg/kg doses of enoxaparin. The results are depicted inFIG. 10 and Table 15.

TABLE 15 Statistical Analysis of the Efficacy Data (Student's t-test).M118- M118- Enoxaparin Enoxaparin Enoxaparin REH REH sodium sodiumsodium Control 0.5 mg/kg 1 mg/kg 2 mg/kg 3 mg/kg 4 mg/kg Mean (min) 11.329.2** 55.3#** 23.2** 36.0** 41.8** St. Dev. 2.3 16.8 12.6 8.3 20.3 20.6*p < 0.05 compared to control group; **p < 0.01 compared to controlgroup; #p < 0.01 compared to enoxaparin sodium 3 mg/kg

Beagle Electrolytic-Induced Femoral Artery Injury Model (Lucchesi'sModel):

The antithrombotic and anticoagulant effects of M118-REH were studied ina canine model of deep arterial thrombosis induced by severeelectrolytic injury.

Surgical Procedures

Animals were premedicated with intramuscular (IM) atropine sulfate (0.02mg/kg) and 1M acepromazine (0.2 mg/kg, ≦3 mg per animal) at least 10-15minutes prior to induction of anesthesia with IV propofol (4-8 mg/kg).Animals were intubated and maintained in anesthesia via isofluraneinhalant, to effect, through a volume-regulated respirator.

A longitudinal incision was made on the medio-ventral surface of theneck to gain access to the tissues overlying the carotid arteries. Onecarotid artery and both jugular veins (one for backup) were subsequentlyexposed for approximately 2 cm by blunt dissection and supported byretaining ligatures at the proximal and distal ends. Two other incisionswere made that initiated on the abdomen and extended distally along thepectineus for a distance covering 66%-75% of each femur. The fascia wasopened at each incision, and the underlying femoral artery was exposedfor a distance of approximately 2-3 cm.

Animal Instrumentation

Twenty four anesthetized beagle dogs were instrumented withintravascular electrodes through the left and right femoral artery wallsand positioned in direct contact with the intima. Each electrode wasconnected to a constant amperage power source, with the cathode placedat a distant subcutaneous site. A stenotic device was positionedimmediately distal to the electrode, a pervascular Doppler flow probewas positioned proximal to the electrode, and a catheter was insertedinto the carotid artery, all of which were connected to a Gould PonemahPhysiological Platform (Linton Instrumentation, Norfolk, UK) forcontinuous monitoring of arterial blood pressure, blood flow, and heartrate. A second catheter was inserted into the jugular vein for bloodsample collection. Finally, an intravenous line was inserted into aperipheral vein for study treatment infusions, and limb leads wereplaced for electrocardiography (measured at Lead II).

Femoral Artery Electrolytic-Injury Model

After instrumentation, each animal received a continuous IV infusion ofvehicle (0.9% sterile saline) for 90 minutes. Fifteen minutes after theinfusion started, electrical current (300 μA) was applied to the rightfemoral artery (control) through the intravascular electrode andadministered continuously until full thrombus formation, defined as areduction in Doppler flow to ≦2% of baseline values, or until the end ofthe observation period at 180 minutes after initiation of electrolyticinjury if the vessel remained patent. Following this, the vessel wasligated proximally and distally to the site of electrolytic injury, thesegment was harvested, and any thrombus, if present, was weighed.

In this model, platelet-rich intravascular thrombi form in proximity toa distal arterial stenosis. Accordingly, the stenotic device wasadjusted to limit hypoxia-induced reactive hyperemia to ≦80% of thebaseline response to physical occlusion (baseline reactive hyperemia wasdetermined at each femoral artery immediately prior to vehicle oractive-treatment infusions). To ensure that hemodynamic properties atthe site of injury approximated normal blood flow through the femoralartery, mean arterial pressure and heart rate were targeted toapproximately 70 mm Hg and 100 beats per minute, respectively, viaisoflurane anesthetic management.

After completing the preceding experiment to evaluate baselinethrombotic parameters in each animal at the right femoral artery,identical procedures were carried out at the left femoral artery(active-treated) in the presence of M118-REH or UFH infusions. Animalswere allocated in 4 treatment groups (n=6). Three groups received IVboluses of M118-REH at 37.5, 75, and 150 anti-Xa IU/kg, respectively,followed by continuous infusions of M118-REH at 1.0 anti-Xa IU/kg/minfor 90 minutes. The fourth group received an IV bolus of UFH at 75 U/kg,followed by a continuous infusion of UFH at 1.0 U/kg/min for 90 minutes.Continuous electrolytic injury was initiated 15 minutes after bolustreatment, and subsequent analyses were carried out identically to thevehicle group.

Assays for cutaneous bleeding time (CBT) and blood collections werecarried out at protocol-specified time points (see below). Physiologicalparameters monitored throughout the procedure included pulse rate,respiration rate, direct blood pressure, rectal temperature, tidalvolume, end-tidal carbon dioxide levels, and O₂ saturation.

Hematology and Coagulation Determinations

For hematology and coagulation determinations, whole blood samples(approximately 100 μL) were collected at 15, 30, 45, 60, 75, 90, 105,120, 135, 150, 165, and 180 minutes for determination of ACT. ACT wasassessed in a HEMOCHRON® Jr. Signature+ Microcoagulation System (ITC,Edison, N.J., USA) according to the manufacturer's instructions. Twoaliquots (approximately 1.3 mL each) of whole blood were collected at 15minutes, 60 minutes, and either 180 minutes or the time of occlusion, ifapplicable, for hematological and further coagulation assays.Hematologic parameters (WBC, RBC, HGB, CHT, MCV, MCH, MCHC, PLT, RTC,ARTC, and WBC differentials) were assayed using an Advia 120 HematologySystem (Bayer Diagnostics Norden, Lyngby, Denmark) following themanufacturer's instructions. Coagulation assays (prothrombin time [PT],activated partial thromboplastin time [aPTT], and fibrinogen levels[FIB]) were conducted using an MLA Electra 1400C Coagulation Analyzer(Beckman Coulter, Fullerton, Calif., USA) following the manufacturer'sinstructions.

To determine anti-Factor Xa and anti-Factor IIa levels, citrated wholeblood samples collected at 0, 15, and 60 minutes after initiation oftreatment were centrifuged at 3,000 g for approximately 10 minutes in arefrigerated centrifuge. Plasma was collected, snap frozen at −20° C.,and stored at −80° C. until ready for testing by chromogenic assay.Stachrome Heparin Anti-Xa kit (Diagnostica Stago, Asnieres sur Seine,France) and a reagent set consisting of substrate 52238, bovinethrombin, and human antithrombin III (Chromogenix, Milano, Italy) wereused for the anti-Factor Xa and anti-Factor IIa assays. All chromogenicassays were quantified in a STA-R Analyzer (Diagnostica Stago) followingMomenta Pharmaceuticals, Inc., SOP for anti-Factor Xa and anti-FactorIIa activity measurements. CBT was determined at 15 minutes, 60 minutes,and either 180 minutes or the time occlusion, if applicable. CBTassessment were carried out at a forelimb by Mielke method using thetest facility's SOP.

Statistics

Values were reported as means±standard deviation (SD) unless otherwisenoted. Means were compared using the Student's t-test assuming equalvariance in different treatment groups. The incidence of occlusion wascompared between treatment groups by calculating the odds ratio relativeto control and using a z test to derive the P-value. Significance levelsfor all tests were set at 0.05.

Anti-Factor Xa and Anti-Factor IIa Plasma Activity

Various doses of M118-REH were compared to a standard dose of UFH (75U/kg). M118-REH exhibited dose-dependent inhibition of both Factor Xaand IIa, with the highest dose of M118-REH (150 IU/kg) demonstratingsimilar anti-Factor Xa activity relative to UFH (FIG. 13 and Table 16).Anti-Factor IIa activity increased proportionally with anti-Factor Xaactivity for both M118-REH and UFH, although the correlation coefficientwas greater for M118-REH (r²=0.890) than UFH (r²=0.465) (FIG. 14).Finally, the ratio of anti-Factor Xa activity to anti-Factor IIa plasmaactivity over time was generally more constant with M118-REH than UFH,consistent with the known variable metabolism of the large andpolydisperse UFH molecules (FIG. 15).

TABLE 16 Summary of Selected Hematologic Endpoints Anti- ACT Factor XaAnti-Factor IIa Treatment (sec)* (IU ± SD)* (IU ± SD)* Control  69 ± 60.0 ± 0.0 0.0 ± 0.0 M118-REH (37.5 IU/kg)^(†) 101 ± 7^(‡) 1.20 ± 0.160.64 ± 0.12 M118-REH (75 IU/kg)^(†) 108 ± 13^(‡) 1.66 ± 0.37 0.71 ± 0.20M118-REH (150 IU/kg)^(†) 141 ± 28^(§) 2.31 ± 0.13 1.06 ± 0.09 UFH (75U/kg)^(†) 163 ± 55^(||) 2.89 ± 1.31 0.95 ± 0.19 ACT, activated clottingtime; IU, international unit; SD, standard deviation; UFH,unfractionated heparin *Recorded at 60 min after initiation of testarticle infusion; ^(†)Bolus dose; ^(‡)P < 0.05, M118-REH vs. UFH; ^(§)P< 0.01, M118-REH vs. control; ^(||)P < 0.01, UFH vs. control.

Coagulation Assays

Next, the anti-coagulant activity of M118-REH was compared to a standarddose of UFH. M118-REH dose-dependent inhibition of clotting was observedwithin 15 minutes in all coagulation assays and was maintained duringthe course of the 60-minute observation period (FIG. 16). At 60 minutes,significantly longer clotting times in the ACT assay were observed afterUFH treatment than M118-REH at 37.5 or 75 anti-Xa IU/kg (Table 16). Thedifference between UFH and M118-REH in the ACT assay was non-significantwhen M118-REH was administered at 150 anti-Xa IU/kg (Table 16). Controlexperiments demonstrated that differences between treatment groups inthe coagulation assays were not attributable to alterations in theconcentrations of the fibrinogen substrate (data not shown).

Antithrombotic Effects

Based on the observation that M118-REH at a dose of 150 anti-Xa IU/kghad similar anticoagulant properties to heparin at a standard dose of 75U/kg, the antithrombotic efficacy of M118-REH at 150 anti-Xa IU/kg, aswell as at lower doses, was compared to UFH in the canine model of deeparterial thrombosis. During infusion of vehicle, full occlusion of thecontrol artery occurred in 24/24 [100%] animals within the observationperiod of 180 minutes (FIG. 17). In the UFH treatment group, 5/6 (83.3%)animals reached the model-defined decrease in Doppler flow. Bycomparison, full occlusion occurred in 3/6 (50%), 2/6 (33.3%), and 1/6(16.7%) animals receiving M118-REH bolus doses of 37.5, 75, and 150anti-Xa IU/kg, respectively, consistent with a dose-responserelationship. The treatment differences between M118-REH- andvehicle-treated arteries for occlusion rates were statisticallysignificant at all M118-REH bolus doses (P<0.05). The difference betweenM118-REH at a bolus dose of 150 anti-Xa IU/kg and UFH was alsosignificant (P<0.05). Thus, M118-REH at 150 anti-Xa IU/kg showedsuperior efficacy to UFH at 75 U/kg, despite the fact that theanticoagulant activity of M118-REH and UFH were comparable at thesedoses. M118-REH at the lower tested doses (37.5 and 75 anti-Xa IU/kg),which were associated with lower anticoagulant activities, hadantithrombotic effects that were more generally similar to UFH.

Mean times to occlusion were 59±25, 132±42, 165±23, 161±41, and 165±36minutes in animals treated with vehicle, UFH, M118-REH (37.5 anti-XaIU/kg), M118-REH (75 anti-Xa IU/kg), and M118-REH (150 anti-Xa IU/kg),respectively. It should be noted that mean occlusion times in theactive-treated arteries were under-estimates of their true values, sincemany arteries, particularly in the M118-REH treatment groups, did notfully occlude by the prespecified endpoint of 180 minutes (such arterieswere arbitrarily assigned an occlusion time of 180 minutes for theanalysis). Mean thrombus weights were 24.0±9.15 mg in thevehicle-treated arteries, 19.4±9.47 mg in the UFH-treated arteries, and24.5±12.17 mg, 19.8±6.24 mg, and 12.8±5.99 mg in the M118-REH-treatedarteries at 37.5, 75, and 150 anti-Xa IU/kg, respectively. Thedifferences between treatment groups for mean time to occlusion and meanthrombus weight did not reach statistical significance.

Cutaneous Bleeding Times

CBT varied from 80±15.5 to 160±52.5 seconds during vehicleadministration at the protocol-specified time points in all groups(Table 17). UFH and M118-REH treatment resulted in minimal increases inCBT, and the effects were highly variable. Group means ranged from135±90.5 to 275±306.8 seconds after UFH treatment and 110±36.3 to190±86.3 seconds after M118-REH administration at all time points.

TABLE 17 Cutaneous Bleeding Times Baseline CBT Femoral CBT (sec ± SD)Treatment (sec ± SD) Artery 15 min 60 min 180 min UFH 100 ± 31.0 Control125 ± 29.5 138 ± 45.5 100 ± 36.3 (75 U/kg) Treated 135 ± 90.5 145 ± 35.1 275 ± 306.8 M118-REH  95 ± 35.1 Control  80 ± 15.5 130 ± 36.3 115 ±58.2 (37.5 IU/kg) Treated 145 ± 64.1 125 ± 48.1 110 ± 36.3 M118-REHControl 130 ± 36.3 130 ± 17.3 160 ± 52.5 (75 IU/kg) 140 ± 24.5 Treated160 ± 45.2 190 ± 86.3 160 ± 31.0 M118-REH Control 115 ± 51.7 110 ± 36.3115 ± 48.1 (150 IU/kg) 110 ± 41.0 Treated 150 ± 50.2 125 ± 64.1 135 ±94.4 UFH, unfractionated heparin, SD, standard deviation, IU,international units, U, units.

Hemodynamic Parameters

No clinically meaningful changes were observed in cardiovascularparameters or clinical chemistry during the course of the experiments.Across the study population, differences between poststenotic hyperemicresponses at the vehicle- and active-treated arteries were ≦13 mL/min inall animals. The goal of limiting hypoxia-induced hyperemic response to≦80% of the baseline response to physical occlusion was met in all but 2arteries: 1 in which the response was 81% (M118-REH [75 anti-Xa IU/kg];control artery); and 1 in which the response was 88% (M118-REH [75anti-Xa IU/kg]; active treatment artery). Mean arterial pressure andheart rate during the course of the procedure in all treatment groupswere 68-78 mm Hg and 96-111 bpm, respectively, close to the 70 mm Hg and100 bpm targets pre-specified in the protocol. The maximum difference inmean arterial pressures and heart rate between the vehicle- andactive-treated arteries in any given animal was 6 mm Hg and 5 beats perminute, respectively (P-values for differences were non-significant).

SUMMARY

M118-REH at 150 anti-Xa IU/kg showed statistically superiorantithrombotic efficacy to a standard dose of UFH (75 U/kg), despite thefact that M118-REH and UFH showed comparable activity in ACT, aPTT, andPT assays at these concentrations. Thus, the incidence of fullthrombus-induced occlusion of the femoral artery was significantly lowerin M118-REH-treated than UFH-treated animals (1/6 [16.7%] vs. 5/6[83.3%], respectively; P<0.05).

The antithrombotic effects observed in the M118-REH treatment groupswere achieved without evidence of complications. No instances ofspontaneous bleeding were documented, and only minimal increases in CBTwere observed at all M118-REH concentrations and experimental timepoints. Additionally, there were no unexpected mortalities or clinicallymeaningful changes in cardiovascular parameters and clinical chemistry.

Two features of the coagulation data in this study were noteworthy.First, M118-REH was measurable in a dose-dependent fashion bypoint-of-care ACT assays. Its ACT response was well correlated toanti-Factor Xa activity compared with UFH. This feature may simplifyadministration of LMWHs in interventional and surgical settings. Second,M118-REH exhibited not only in vitro anti-Factor Xa activity, but alsosignificant anti-Factor IIa activity, a characteristic that furtherdistinguishes M118-REH from currently available LMWH options (J. Hirshet al. “Heparin and low-molecular-weight heparin: mechanisms of action,pharmacokinetics, dosing, monitoring, efficacy, and safety,” Chest.2001; 119:64 S-94S). Enoxaparin, for instance, is characterized by aratio of anti-Factor Xa activity to IIa activity of 17.2; by comparison,the analogous ratio for UFH is 3.3 (U. Cornelli and J. Fareed. “Humanpharmacokinetics of low molecular weight heparins,” Semin Thromb Hemost.1999; 25 Suppl 3:57-61). In this study, M118-REH had a ratio ofanti-Factor Xa to IIa activity that was approximately 2-2.5 (FIG. 15),demonstrating enhanced anti-Factor IIa activity relative to other LMWHs.The ratio was generally more consistent over time than UFH, as predictedby the known variable metabolism of the large and polydisperse UFHmolecules.

TABLE 18 Summary Table of Thrombotic and Selected Hematologic EndpointsThrombus Anti- Anti- Weight TTO ACT Factor Factor Treatment Occlusion(%) (mg) (min)^(a) (sec)^(b) Xa^(b) IIa^(b) Control (RFA) 24/24 (100%)24.0 ± 9.2 59 ± 25 69 ± 6^(c) 0.00 ± 0.00 0.00 ± 0.00 UFH (Grp 1) 5/6(83%) 19.4 ± 9.5 132 ± 42^(e) 163 ± 55^(e) 2.89 ± 1.31 0.95 ± 0.19 M118(Grp 4) 3/6 (50%)^(c)  24.5 ± 12.2 165 ± 23^(e) 101 ± 7^(d)  1.20 ± 0.160.64 ± 0.12 M118 (Grp 2) 2/6 (33%)^(c) 19.8 ± 6.2 161 ± 41^(e) 108 ±13^(d) 1.66 ± 0.37 0.71 ± 0.20 M118 (Grp 3) 1/6 (17%)^(c,d)  12.8 ±6.0^(e) 165 ± 36^(e) 131 ± 16  2.31 ± 0.13 1.06 ± 0.09 TTO = Time toocclusion; ACT = Activated clotting time; RFA = Right femoral artery;UFH = unfractionated heparin; Grp = Group. ^(a)TTO >180 min was set at180 min. ^(b)Value recorded at 60 min after initiation of test articleinfusion. ^(c)P < 0.05 vs. control. ^(d)P < 0.05 vs. UFH. ^(e)P < 0.01vs. control.In vivo Neutralization:

In vivo studies were performed employing Sprague-Dawley rats and NewZealand rabbits. M118-REH, enoxaparin or two unfractionated heparins(UFH) were administered intravenously at different doses at t=0.Neutralization of the pharmacologic effects of each of these treatmentswere evaluated by tail vein injection of protamine sulfate 5 minutesafter t0 at ratios of 0.5 and 1 mg to 100 anti-Xa IU (or 100 USP Unit or1 mg in case of UFH). Blood samples were obtained and tested for anti-Xaand anti-IIa activity at baseline (just prior to protamine injection) 5,30 and 60 minutes post-protamine sulfate administration.

Complete and rapid neutralization of M118-REH anti-Xa activity byprotamine sulfate in vivo was achieved at ratios of 0.5 mg: 100 anti-XaIU (>98%) in rats and 1 mg:100 anti-Xa IU (more than 99% in rats and 95%in rabbits) at 5 minutes-post intravenous protamine sulfate delivery.There was no “rebound” of anti-Xa activity observed within 1 hour postprotamine sulfate administration.

Neutralization of anti-Xa activity was comparable between M118-REH andUFH at ratios of 0.5 mg and 1 mg to 100 anti-Xa IU (or 100 USP or 1 mgUFH). Greater than 40% and 20% of the anti-Xa activity remained 5minutes after protamine sulfate injection in rats dosed with enoxaparinat ratios of 0.5 and 1 mg:100 anti-Xa IU, respectively. Approximately38% of the anti-Xa activity remained in rabbits administered enoxaparinat 1 mg:100 anti-Xa IU protamine sulfate. More than 90% of the anti-IIaactivity following each of the heparins was neutralized at ratios of 0.5and 1 mg to 100 anti-Xa IU. The magnitude of reversal of anti-IIaactivity by protamine sulfate was equivalent among the three treatments,i.e. M118-REH, enoxaparin or UFH. The results are depicted in FIG. 11.

In Cynomologus monkeys, protamine sulfate (PS) reversed both M118-REHanti-Xa and anti-IIa activity at a similar extent in a dose dependentfashion. M118-REH was administered intravenously to consciouscynomologus monkeys, in some cases followed by administration of PS.Blood samples were obtained and evaluated for M118-REH concentration asmeasured by anti-Xa and anti-IIa activity and coagulation profile.Hematology, cutaneous bleeding times and signs of clinical toxicity weremonitored for 24 hours.

M118-REH activity was detectable immediately following iv administrationat a dose of 150 anti-Xa IU/kg. First order elimination kinetics forM118-REH was observed with t1/2 of 0.50±0.04 hr (anti-Xa) and 0.68±0.05hr (anti-IIa). Furthermore, PS rapidly reversed M118-REH anti-Xa andanti-IIa activity in a dose-dependent manner. The majority of anti-Xa(93.6±1.2%) and anti-IIa (90.14±1.2%) activity was rapidly neutralizedby PS at a ratio of 1.5 mg PS per 100 IU anti-Xa activity, the higherdose studied. ACT and aPTT measurements closely correlated with anti-Xaactivity (r2=0.95 and 0.99, respectively). M118-REH increased ACT 2-3times and was reversed to baseline values within 5 minutes followingintravenous PS administration. No signs of clinical toxicity or adversebleeding were observed.

Administration of M118-REH causes a consistent and rapid anticoagulantaffect that can be rapidly reversed by injection of PS. M118-REHanticoagulation is easily measured and monitored by ACT and aPTT. Thereversibility and monitorability of M118-REH are unique compared withcompared with other commercially available LMWHs.

The results of neutralization studies of M118-REH in the NHP model arepresented in Tables 19-21 below.

TABLE 19 Neutralization of M118-REH in the NHP Model - Most aPTT and ACTCame Back to Normal After Dosing. M118-REH{circumflex over ( )}M118-REH{circumflex over ( )} M118-REH{circumflex over ( )}{circumflexover ( )} UFH{circumflex over ( )}{circumflex over ( )} 1 mg: 1 mg: 1001.5 mg: 100 2 mg: 100 100 IU Anti-Xa IU Anti-Xa IU Anti-Xa IU ACTBaseline 99.5 ± 9.2 85.3 ± 4.2 93.0 ± 5.1 97.0 ± 4.2 Post-M118- 168.5 ±14.9 174.3 ± 12.3 193.0 ± 9.7  139.0 ± 21.2 REH 5′ Post  97.5 ± 14.9103.0 ± 4.4  99.2 ± 7.5 89.5 ± 0.7 Protamine (NS) (NS) (NS) aPTTBaseline 31.95 ± 0.1  23.4 ± 2.7 20.4 ± 0.9 26.1 ± 0.8 Post-M118-  46.9± 31.9†  177.9 ± 59.0*  162.6 ± 85.5* 48.1 ± 1.8 REH 5′ Post 24.2 ± 0.432.5 ± 4.4 29.6 ± 6.0 25.9 ± 1.6 Protamine (NS) (NS) NS: no significantdifference compared to UFH *aPTT >212 sec in some samples; †½ lower thanbaseline after UFH dosing {circumflex over ( )}M118-REH dosed at 150IU/kg i.v. {circumflex over ( )}{circumflex over ( )}M118-REH dosed at75 IU/kg, UFH dosed at 75 IU/kg

TABLE 20 Neutralization of M118-REH in the NHP Model - SignificantAnti-Xa/IIa Activities Remained M118-REH M118-REH M118-REH UFH 1 mg: 1mg: 100 1.5 mg: 100 2 mg: 100 100 IU anti-Xa IU anti-Xa IU anti-Xa IUAnti-Xa Baseline 0.3 ± 0.4 0.0 ± 0.0 0.0 ± 0.0 n/a Post-M118- 2.0 ± 0.33.6 ± 0.4 6.1 ± 0.8 1.2 ± 0.1 REH 5′ Post 0.1 ± 0.1 0.5 ± 0.0 0.4 ± 0.10.2 ± 0.1 Protamine Anti0-IIa Baseline 0.4 ± 0.4 0.0 ± 0.1 0.0 ± 0.0 n/aPost-M118- 1.9 ± 0.4 2.1 ± 0.3 3.0 ± 1.2 1.6 ± 0.0 REH 5′ Post 0.1 ± 0.10.3 ± 0.1 0.3 ± 0.1 0.4 ± 0.2 Protamine

TABLE 21 Neutralization of M118-REH in the NHP Model - Percent ofReduction for Different Parameters After Protamine M118-REH M118-REHM118-REH UFH 1 mg: 1 mg: 100 1.5 mg: 100 2 mg:: 100 100 IU anti-Xa IUanti-Xa IU anti-Xa IU ACT 41.5 ± 14.0 40.8 ± 2.2 47.7 ± 1.2 34.9 ± 9.4 aPTT 32.4 ± 46.9 80.6 ± 5.4  74.8 ± 16.2 46.0 ± 16.9 Anti-Xa 97.3 ± 3.8 85.2 ± 2.0 93.6 ± 1.2 84.0 ± 9.1* Anti-IIa 97.2 ± 3.9  83.4 ± 4.2 90.1 ±1.2  75.1 ± 10.1* †equation: (post-M118-REH -post-protamine)*100/post-M118-REH *assay instable

In Vivo Bedside Monitoring Study in Preclinical Models:

M118-REH, enoxaparin and unfractionated heparin were administratedintravenously at different doses in rats or rabbits. Blood samples wereobtained for ACT measurement and anti-Xa test. Hemachron Junior and ACTplus cuvette were used for ACT measurement while anti-Xa was measuredwith Coag-A-Mate MTX II. The correlation between anti-Xa and ACT wascompared among these three heparins. In New Zealand White rabbits, 1mg/kg M118-REH and UFH were injected via ear marginal vein. Bloodsamples were collected at 5′, 15′, 30′ 1, 2, 3, 4 hrs after heparindelivery. In Sprague-Dawley rats, M118-REH and UFH were dosed at 0.5 and1 mg/kg while enoxaparin dosed at 1 mg/kg and 2 mg/kg. Those dosesachieved significant anti-Xa and ACT elevation both in rats and rabbits.For M118-REH, the correlation factor (r²) of anti-Xa to ACT was 0.79 inrabbits and 0.85 in rats. The correlation factors (r²) between anti-Xaand ACT for UFH were 0.31 in rabbits and 0.79 in rats, while forenoxaparin it was only 0.66 in rats. The results are show in FIG. 12.

In NHP models, after intravenous injection, M118-REH presented firstorder of elimination with half life as 0.50±0.04 or 0.68±0.05 hour byanti-Xa and anti-IIa measurement, respectively. Distribution volumes(Vd) are 32.01±2.21 (anti-Xa) or 48.58±0.95 mL/kg (anti-IIa,)respectively. Clearance (Cl) are 37.24±4.65 (anti-Xa) and 43.92±5.38(anti-IIa) mL/hr/kg. The ACT and aPTT results are closely correlated toanti-Xa activity (correlation ratio r=0.95 and 0.99 respectively).

In Vivo Hemorrhage Test:

Low molecular weight heparin is generated by depolymerization ofunfractionated heparin with different chemical or enzymatic processes.Bleeding time measurements have been frequently employed in thedevelopment of new antithrombotics as an indication for risk ofbleeding. This objective of this study was to investigate the risk ofbleeding by standard bleeding time measurement for M118-REH and comparethe risk to that posed by comparable treatments such as enoxaparin andunfractionated heparin.

M118-REH, enoxaparin and unfractionated heparin were administratedintravenously as a single bolus dose of 0.5 mg/kg via a marginal earvein in rabbits. Bleeding time (BT) measurements were made on the ear atbaseline and 5, 15, 30, 60, 120 and 180 minutes after test articleadministration. M118-REH caused a 3-4 fold increase in BT at 5 minutescompared to 60 minutes post-administration (p<0.05). A similar responsein BT was observed by treatment with enoxaprin and UFH. Bleeding timesreturned to within normal range by 120 minutes and remained nearbaseline values for the remaining time points. No other adverse clinicalfindings were observed in any of treatment groups at the dose tested.

As shown in Table 22, in NHP models, M118-REH caused prolongation ofCurtis bleeding time (CBT) after intravenous injection, there is nostatistically significant difference from baseline (p<0.05).

TABLE 22 CBT of M118-REH after intravenous injection at 150 anti-XaIU/kg in the NHP model. Time point 0 0.25 1 1.5 (hour) Bleeding time 1.3± 0.6 2.5 ± 0.7 2.5 ± 1.0 2.5 ± 1.3 (minutes) NS NS NS

In beagles, UFH produced longer CBT than M118-REH after bolus injectionand continuous infusion.

In Vivo Coagulation System:

Platelet Interaction:

In NHP models, there was no observation on the influence on plateletaggregation triggered by ADP after M118-REH bolus intravenousadministration.

Fibrinolytic Pathway Intervention:

In NHP models, as shown in Table 18 below, after M118-REH intravenousbolus injection, fibrinogen level was consistent and there was nostatistical significant difference from baseline.

TABLE 23 Fibrinogen and Prothrombin time (PT) after M118-REH intravenousinjection at 150 anti-Xa IU/kg in the NHP model. Prothrombin TimeFibrinogen (mg/dL) (second) baseline 174.3 ± 35.0 11.4 ± 0.5  5′ 168.3 ±28.0  16.0 ± 0.9** 30′ 166.0 ± 30.5  13.8 ± 0.9* 60′ 172.0 ± 29.7  14.1± 1.1* 90′ 170.0 ± 27.9 12.8 ± 2.2 360′  163.7 ± 29.5 11.4 ± 0.9 360′ 164.3 ± 27.0 10.8 ± 0.3 1440′  176.0 ± 2.0  10.7 ± 0.3

Multiple Ascending Dose Studies

In repeat dose IV studies in the rat, increase in exposure to M118appeared to be proportional to the increase in dosage on study Days 0and 13. In male rats, exposure to M118 in terms of total anti-Xaactivity increased from Day 0 to Day 13, but remained similar in termsof total anti-IIa activity. In female rats, exposure to M118 decreasedfrom Day 0 to Day 13. Female rats appeared to have higher exposure toM118 than male rats on Day 0, but on Day 13 exposures to M118 weresimilar between genders. In repeat dose IV studies in the dog, systemicexposure to M118 increased as dosage increased over the 5 to 50mg/kg/day range. However exposure to M118 generally did not increaseproportionally with increasing M118. There was no evidence ofaccumulation over the 14 day period. Half-lives for anti-Xa and anti-IIaactivity in dog plasma ranged from 1.0 to 3.2 hours with no particulartrend related to dosage or gender. The half-lives tended to be shorteron Day 13 than on Day 0. There was no consistent trend related to M118dosage with respect to the apparent systemic clearance or the apparentvolume of distribution of anti-Xa and anti-IIa activities.

1. A low molecular weight heparin (LMWH) composition comprising:oligosaccharide chains that have the following structure:

and oligosaccharide chains that have the following structure:

wherein R is H or SO₃Na; R₁ is SO₃Na or COCH₃; n=2-50; wherein thecomposition has the following properties: (a) an average value for n of9-16, (b) 5 to 15% ΔUH_(NAc,6S)GH_(NS,3S,6S) as measured by mole %, aweight average molecular weight of 5500 to 8500 Da, (d) anti-Xa activityof 120 to 380 IU/mg, (e) an anti-Xa to anti-IIa ratio of 2:1 to 1:1, and(f) the ratio of (e) is constant over a period of about 30 to 120minutes after administration to a subject. 2-29. (canceled)
 30. Apharmaceutical composition comprising a LMWH composition of claim 1 anda pharmaceutically acceptable carrier.
 31. A method of treating asubject having or at risk of having a thrombolytic disorder, comprisingadministering a composition of claim 1 to a subject, to thereby treatthe disorder. 32.-47. (canceled)
 48. A method of monitoring a subjecttreated with a LMWH composition of claim 1 comprising: evaluatingactivated clotting time (ACT) or activated partial thromboplastin time(aPTT) in a subject who has been administered a LMWH composition ofclaim 1
 49. A method of treating a subject who has been administered aLMWH composition of claim 1 comprising: administering protamine sulfateto a subject to neutralize some or all of the anti-Xa activity, anti-IIaactivity or both of a LMWH composition of claim 1 in the subject. 50.(canceled)
 51. A method of making a low molecular weight heparin (LMWH)composition, the method comprising providing a precursor LMWHcomposition having an average chain length of about 8 to 14disaccharides obtained by salt precipitation and cleavage of a heparinsample with an enzyme which cleaves at unsulfated uronic acid linkages;and subjecting the precursor LMWH composition to a size selection stepto obtain a LMWH composition having an average chain length (n) of about9 to 16 disaccharides, wherein the LMWH composition comprises:oligosaccharide chains that have the following structure:

and oligosaccharide chains that have the following structure:

wherein R is H or SO₃Na; R₁ is SO₃Na or COCH₃; n=2-50; and wherein thecomposition has the following properties: (g) 5 to 15%ΔUH_(NAc,6S)GH_(NS,3S,6S) as measured by mole %, (h) weight averagemolecular weight of 5500 to 8500 Da, (i) anti-Xa activity of 120 to 380IU/mg, (j) an anti-Xa to anti-IIa ratio of 2:1 to 1:1, and (k) the ratioof (d) is constant over a period of about 30 to 120 minutes afteradministration to a subject. 52.-57. (canceled)
 58. The method of claim51, wherein the size selection step comprises chromatography orfiltration.
 59. The method of claim 58, wherein the chromatography issize-selection chromatography or ion-exchange chromatography.
 60. Themethod of claim 51, wherein the enzyme is a heparinase III.
 61. Themethod of claim 51, wherein the enzyme is a modified heparinase IIIenzyme having a substitution of an alanine for a histidine at His225.62. The method of claim 51, wherein the enzyme is heparin sulfateglycosaminoglycan lyase III from Bacteroides thetaiotaomicron.
 63. Themethod of claim 51, wherein the LMWH composition has an averagemolecular weight of about 5700 to 7900 Da.
 64. The method of claim 51,wherein the LMWH composition has an anti-Xa activity of about 170 to 330IU/mg.
 65. The method of claim 64, wherein the LMWH composition has ananti-Xa activity of about 180 to 300 IU/mg.
 66. The method of claim 51,wherein the LMWH composition has an anti-IIa activity of about 130 to190 IU/mg.
 67. The method of claim 66, wherein the LMWH composition hasan anti-IIa activity of about 155 to 185 IU/mg.
 68. The method of claim51, wherein the LMWH composition has less than 1000 ng/mg of aheparinase enzyme; less than 1% w/w methanol; less than 1% w/w ethanol;and less than 2000 ppm free sulfate.
 69. The method of claim 51, wherein15 to 35% of the total number of chains in the composition have a Δ4,5unsulfated uronic acid at the non-reducing end.
 70. The method of claim51, wherein at least 60% of the chains in the composition have anN-acetylated hexosamine at the reducing end.
 71. The method of claim 70,wherein at least 80% of the chains of the composition have anN-acetylated hexosamine at the reducing end.
 72. The method of claim 51,further comprising precipitating the precursor LMWH composition havingan average chain length of about 8 to 14 disaccharides prior to the sizeselection step.
 73. The method of claim 51, further comprisingprocessing the LMWH composition into drug product.